Mechanistic Insights Into the PARP10-Mediated ADP

Mechanistic insights into the PARP10-mediated

ADP-ribosylation reaction and

analysis of PARP10’s subcellular localization

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologe

Henning Schuchlautz

aus Waldbröl

Berichter: Universitätsprofessor Dr. Bernhard Lüscher Privatdozent Dr. Christoph Peterhänsel

Tag der mündlichen Prüfung: 20. Juni 2008

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. I

Abstract

ADP-ribosylation controls many cellular processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARPs 6-16, raising questions about how these enzymes function. Therefore the enzymatic properties of PARP10, a representative of the novel PARP enzymes, were analyzed in detail. It was found that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has mono- ADP-ribosyltransferase rather than polymerase activity. Despite this fundamental difference PARP10 also modifies acidic residues. Molecular modeling shows that an acidic residue of the substrate can be placed in a favorable position to stabilize the oxocarbenium transition state of the reaction. This function is normally executed by the catalytic glutamate in ADP-ribosyltransferases and bona fide PARP enzymes. Consequently, a novel catalytic mechanism for PARP10 is proposed, in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate, using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. Accordingly, it is suggested to subdivide the PARP family into three classes: one representing bona fide PARP enzymes with poly-ADP-ribose polymerase activity like PARP1, a second one representing PARP-related mono-ADP-ribosyltransferases like PARP10, and a third one without enzymatic activity due to the lack of critical residues in the NAD+ binding fold. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells. The transport across the nuclear envelope through nuclear pore complexes is achieved by the interaction of cargo proteins with karyopherins that mediate transport processes into and out of the nucleus. These interactions are in general dependent on the presence of a nuclear localization signal (NLS) or a nuclear export sequence (NES) in the cargo protein. It was shown previously that PARP10 possesses a functional NES and accumulates in the nucleus if nuclear export is inhibited. Since PARP10’s molecular weight exceeds the diffusion limit of the nuclear pore complexes significantly, it has to be imported into the nucleus in an active fashion. In order to locate a potential NLS in PARP10 subcellular localization studies and fluorescence bleaching techniques were performed to visualize nucleocytoplasmic shuttling. While it was possible to confirm the presence of a single classical NES in PARP10, no classical NLS could be II detected. In fact the data suggest that three regions within PARP10 have the ability to enter the nucleus and thus potentially contribute to the nuclear import of PARP10. Surprisingly, the analysis of the subcellular PARP10 localization revealed the enrichment in remarkable cytoplasmic foci. Co-localization studies showed no conclusive overlap between PARP10 foci and previously described cytoplasmic substructures. Using time-lapse microscopy it was possible to show that PARP10 foci are highly dynamic, tend to fuse, can disintegrate and form de novo. Importantly, the deletion of the N-terminal RNA recognition motif in PARP10 dramatically impaired the formation of these foci indicating a potential role for RNA in their development. Although PARP10 foci do not overlap with RNA-processing particles, i.e. P bodies or stress granules, certain components present in P bodies are found in these foci. This co-localization is greatly enhanced in the presence of stress that blocks translation and triggers the formation of stress granules. Thus PARP10 enriches in novel cytoplasmic foci that might play a role in RNA processing suggesting a role for PARP10 in the regulation of cellular stress responses.

III

Zusammenfassung

Viele zelluläre Prozesse werden mittels ADP-ribosylierung gesteuert, unter anderem Transkription, DNA-Reparatur und bakterielle Toxizität. ADP-Ribosyl-Transferasen und Poly-ADP-Ribose-Polymerasen (PARPs) katalysieren die Mono- beziehungsweise Poly-ADP-Ribosylierung von Proteinen in Abhängigkeit von einem hoch konservierten Glutamat-Rest in ihrem aktiven Zentrum. Allerdings fehlt dieses Glutamat offensichtlicher Weise in den kürzlich beschriebenen PARPs 6-16, was zu der Frage führt, wie diese Enzyme katalytische ADP-Ribosylierung bewerkstelligen. Um dieser Fragestellung nachzugehen, wurden die katalytischen Eigenschaften von PARP10, einem Vertreter dieser neuen PARP-Enzyme, eingehend untersucht. Anhand dieser Studien lässt sich schlussfolgern, dass PARP10 im Gegensatz zu PARP1 das katalytische Glutamat fehlt and daher als Mono-ADP-Ribosyl-Transferase und nicht als Polymerase fungiert. Trotz dieses fundamentalen Unterschiedes modifiziert PARP10 ebenso wie PARP1 saure Aminosäure-Reste. Anhand eines molekularen Modells der katalytischen Domäne von PARP10 konnte gezeigt werden, dass ein saurer Rest des Substrates so im aktiven Zentrum positioniert werden kann, dass er in der Lage wäre den Übergangszustand der Reaktion, welcher durch ein Carbokation gekennzeichnet ist, zu stabilisieren. Diese Stabilisierung erfolgt normalerweise durch das katalytische Glutamat. Infolgedessen wird ein neuartiger katalytischer Mechanismus für PARP10 vorgeschlagen, in dem der saure Aminosäure-Rest des Substrates funktionell das katalytische Glutamat ersetzt. Die ADP-Ribosylierung durch PARP10 stellt somit eine substrat-assistierte Katalyse dar. Dieser Mechanismus liefert eine Erklärung für die nicht vorhandene Polymerase-Aktivität der neuen PARP-Enyzme. Daher wird die Unterteilung der PARP-Familie in drei Klassen vorgeschlagen: eine mit bona fide PARP-Enyzmen wie PARP1, die Polymerase-Aktivität besitzen, eine weitere mit PARP-ähnlichen Mono-ADP-Ribosyl-Transferasen wie PARP10 und eine dritte mit Proteinen, die aufgrund des Verlustes essentieller Reste in der NAD+-Bindungstasche enzymatisch inaktiv sind. Die Entdeckung mono-ADP-ribosylierender PARP-Enyzme wird hilfreich sein, um die verschiedenen biologischen Funktionen der Mono- gegenüber Poly-ADP-Ribosylierung genauer zu untersuchen. Der Transport über die Kernmembran durch die Kernporen wird erreicht durch Interaktion der zu transportierenden Proteine mit Karyopherinen, welche Import in den und Export aus dem Nukleus vermitteln. Diese Interaktionen sind in der Regel abhängig von einem Kern-Lokalisations-Signal (NLS) oder einer Kern-Export-Sequenz (NES) im zu transportierenden Protein. Es konnte bereits gezeigt werden, dass PARP10 ein funktionelles NES besitzt und nach Inhibition des Kern-Exports im IV

Nukleus zu finden ist. Da das Molekulargewicht von PARP10 die Grenze für freie Diffusion über die Kernporen bei Weitem überschreitet, muss es aktiv in den Nukleus transportiert werden. Um ein potentielles NLS in PARP10 zu identifizieren, wurden subzelluläre Lokalisationen untersucht und Fluoreszenz-Bleich-Techniken angewendet, um nukleozytplasmatischen Transport sichtbar zu machen. Während es möglich war die Gegenwart eines einzelnen funktionellen NES in PARP10 zu bestätigen, konnte ein klassisches NLS nicht nachgewiesen werden. Die vorliegenden Daten lassen vielmehr vermuten, dass drei Bereiche innerhalb von PARP10 die Fähigkeit haben in den Nukleus zu gelangen und daher potentiell zum Kern-Import von PARP10 beitragen. Die Untersuchung der subzellulären Lokalisation von PARP10 zeigte überraschender Weise eine Anreicherung in auffälligen zytoplasmatischen Foci. Kolokalisations- Studien zeigten keine schlüssige Überlappung mit zuvor beschriebenen zytoplasmatischen Substrukturen. Mittels Zeitraffer-Mikroskopie konnte gezeigt werden, dass PARP10-Foci hoch dynamisch sind, dazu neigen zu fusionieren, sich auflösen und wieder de novo bilden können. Bedeutsamer Weise beeinträchtigt die Deletion des RNA-Erkennungs-Motivs in PARP10 die Entstehung dieser Foci merklich, was auf eine Rolle von RNA in deren Bildung hindeuten könnte. Obwohl PARP10-Foci keine Überlappung mit RNA-prozessierenden Partikeln, d.h. P bodies und stress granules, zeigen, kommen bestimmte Komponenten von P bodies auch in PARP10- Foci vor. Diese Kolokalisation kann durch die Erzeugung von Stress, welcher die Translation blockiert und die Bildung von stress granules fördert, deutlich gesteigert werden. Es lässt sich schlussfolgern, dass sich PARP10 in bis dato neuartigen zytoplasmatischen Foci anreichert, die eine Rolle bei RNA-Prozessierung spielen könnten, was auf eine mögliche Funktion von PARP10 in der zellulären Stress-Antwort hindeutet.

Table of Contents

ABSTRACT...... I ZUSAMMENFASSUNG ...... III TABLE OF CONTENTS...... V 1. INTRODUCTION ...... 1

1.1 POST-TRANSLATIONAL MODIFICATIONS: REGULATORS OF PROTEIN FUNCTION ...... 1 1.2 ADP-RIBOSYLATION: AN UNUSUAL POST-TRANSLATIONAL MODIFICATION ...... 3 1.3 THE PARP SUPERFAMILY OF ADP-RIBOSYLATING ENZYMES...... 3 1.3.1 PARP1 ...... 5 1.3.2 PARP2 ...... 14 1.3.3 PARP3 ...... 14 1.3.4 PARP4 ...... 15 1.3.5 PARP5a and PARP5b (Tankyrase1 and 2)...... 15 1.3.6 PARP7 (TiPARP) ...... 17 1.3.7 PARP9, 14, 15 (BAL1, 2, 3 / macroPARPs)...... 18 1.3.8 PARP13 (ZAP(L))...... 19 1.3.9 PARP10 ...... 20 1.4 ADP-RIBOSE BINDING MOTIFS ...... 22 1.5 INSIGHTS INTO THE CATALYTIC MECHANISM OF ADP-RIBOSYLATION REACTIONS ...... 25 1.6 PROTEIN SORTING: DIRECTING PROTEINS TO THE DESIRED DESTINATIONS ...... 36 1.7 TRANSPORT ACROSS THE NUCLEAR ENVELOPE ...... 36 1.8 AIM OF THIS WORK...... 40 2. RESULTS AND DISCUSSION...... 41

2.1 THE PARP10-MEDIATED ADP-RIBOSYLATION REACTION ...... 41 2.1.1 PARP10 functions as mono-ADP-ribosyltransferase ...... 41 2.1.2 Molecular base for the transferase activity of PARP10 ...... 46 2.1.3 Characterization of the catalytic activity of PARP10:...... 50 ADP-ribosylation of acidic residues ...... 50 2.1.4 Identification of Glu882 as auto-ADP-ribosylation site ...... 55 2.1.5 Substrate-assisted catalysis by PARP10 ...... 58 2.1.6 Functional relevance of the conserved loop region between ß-sheets 4/5...... 62 2.1.7 Post-translational regulation of the catalytic PARP10 activity...... 66 2.1.8 Biological significance of the PARP10-mediated ADP-ribosylation reaction ...... 69 2.2 PARP10’S SUBCELLULAR LOCALIZATION AND NUCLEOCYTOPLASMIC SHUTTLING ...... 72 2.2.1 PARP10 fragments display a heterogeneous subcellular localization ...... 72 2.2.2 The nucleocytoplasmic shuttling of PARP10 fragments...... 78 2.2.3 The nucleocytoplasmic shuttling of full-length PARP10 ...... 81 2.2.4 PARP10 enriches in dynamic cytoplasmic foci ...... 86 2.2.5 The N-terminal domains in PARP10 participate in foci formation...... 89 2.2.6 PARP10 foci seem to represent novel cytoplasmic bodies...... 92 2.2.7 PARP10 foci might be related to RNA-processing particles ...... 94 3. CONCLUSIONS ...... 98 4. EXPERIMENTAL PROCEDURES...... 101

4.1 CONSUMABLES AND REAGENTS...... 101 4.2 OLIGONUCLEOTIDES ...... 101 4.3 PLASMIDS...... 103 4.4 ANTIBODIES...... 109 4.5 WORK WITH NUCLEIC ACIDS...... 111 4.5.1 DNA preparation...... 111 4.5.2 RNA preparation...... 111 4.5.3 Enzymatic manipulation of plasmid DNA...... 111 4.5.4 Agarose gel electrophoresis ...... 111 4.5.5 Gel extraction of DNA ...... 112 VI

4.5.6 Gateway cloning ...... 112 4.5.7 Site-directed mutagenesis ...... 112 4.5.8 Generation of pSuper-based siRNA constructs ...... 112 4.6 WORK WITH PROKARYOTIC CELLS ...... 112 4.6.1 Bacteria strains...... 112 4.6.2 Materials for work with prokaryotic cells ...... 113 4.6.3 Protocols for work with prokaryotic cells...... 113 4.6.3.1 Bacterial Transformation...... 113 4.6.3.2 Purification of GST fusion proteins ...... 113 4.6.3.3 Purification of His-tagged TEV protease ...... 114 4.7 WORK WITH EUKARYOTIC CELLS (CELL CULTURE)...... 115 4.7.1 Eukaryotic cell lines...... 115 4.7.2 Materials for cell culture work...... 116 4.7.3 Protocols for work with eukaryotic cells...... 116 4.7.3.1 Cryo-conservation and thawing of cells...... 116 4.7.3.2 Transient transfection ...... 117 4.7.3.3 Preparation of cell lysates ...... 118 4.7.3.4 Colony formation assay ...... 118 4.8 WORK WITH PROTEINS ...... 118 4.8.1 Denaturing Discontinuous Polyacrylamide Gel Electrophoresis (SDS-PAGE)...... 118 4.8.2 Western Blot ...... 119 4.8.3 Immunodetection of proteins ...... 119 4.8.4 Rapid Coomassie Staining ...... 120 4.8.5 Mass-spectrometric analysis ...... 120 4.8.6 Tandem Affinity Purification...... 121 4.9 ENZYMATIC ASSAYS...... 122 4.9.1 PARP assay...... 122 4.9.2 Analysis of ADP-ribosylation reaction products ...... 122 4.9.3 PARG/ARH assay ...... 123 4.9.4 Phosphatase assay...... 124 4.10 MICROSCOPY TECHNIQUES...... 124 4.10.1 Immunocytochemistry ...... 124 4.10.2 Time-lapse microscopy ...... 125 4.10.3 iFLAP (inverted fluorescence localization after photobleaching)...... 125 4.11 MOLECULAR MODELING ...... 125 5. REFERENCES ...... 127 6. APPENDIX...... 150

6.1 ABBREVIATIONS...... 150 6.2 CURRICULUM VITAE ...... 155 6.3 VERÖFFENTLICHUNGEN...... 156 6.3 EIDESSTATTLICHE ERKLÄRUNG ...... 157 6.4 DANKSAGUNG ...... 158

Introduction 1

1. Introduction

1.1 Post-translational modifications: regulators of protein function Proteins are subject to a whole array of post-translational modifications (PTMs) that fulfill regulatory functions on the modified proteins. A brief survey of reversible protein modifications shall illustrate how PTMs regulate protein and thereby cell function by a complex network of agonistic and antagonistic enzymes as well as protein domains serving as readout system and redirecting the initial event to downstream target proteins.

The best-studied example of PTMs is phosphorylation which involves the transfer of the -phosphate group from ATP to specific amino acids; in eukaryotes, these are usually hydroxyl groups of serine, threonine or tyrosine residues. The first protein kinase activity was reported in 1954 (Burnett and Kennedy, 1954) for the phosphorylation of casein. In the next decades, a rapidly growing body of evidence underlined the importance of reversible phosphorylation as a versatile tool to regulate protein function. Phosphatases removing the phosphate group from the modified residues were discovered and were shown to have specificity towards the individual phosphorylated residue (Barford et al., 1998). In addition, a multitude of modular protein domains that recognize phosphorylated amino acid residues have been analyzed in great detail. To name just two examples, SH2 domains and PTB domains recognize phosphotyrosine residues in target proteins achieving binding specificity by their different requirements for adjacent residues (Pawson et al., 2001; Yan et al., 2002).

To date, besides phosphorylation a variety of further PTMs have been identified and are studied including methylation, ubiquitination, acetylation and ADP-ribosylation. Especially in the field of epigenetics, the careful analysis of chromatin modifications has revealed a complex, well coordinated regulation of the chromatin state by PTMs occurring on histone tails (Kouzarides, 2007a) (Figure 1). At the beginning of deciphering the functional relevance of these modifications, the so-called histone code (Strahl and Allis, 2000), it was believed that certain histone marks would be predictive of gene activation while others would correlate with gene repression. However, recent observations suggest that many histone marks are highly dynamic (Berger, 2007). It turns out that many of the histone marks seem to have several, sometimes opposing roles. One modification that has drawn broad attention is the trimethylated lysine of Introduction 2 histone H3 (H3K4me3), a modification that is associated with and regarded as a hallmark of active promoters (Li et al., 2007). Recently, it was shown that the basal transcription factor TFIID directly binds to the H3K4me3 mark in promoter regions resulting in transcriptional activation (Vermeulen et al., 2007). Interestingly, the asymmetric dimethylation of H3R2 (H3R2me2a) inhibits binding to H3K4me3, whereas acetylation of H3K9 and H3K14 potentiates TFIID interaction (Vermeulen et al., 2007). These findings fit with the observation that H3R2me2a counter-correlates with H3K4me3 on human as well as yeast promoters (Guccione et al., 2006; Kirmizis et al., 2007). Recent studies suggest that the H3R2me2a prevents trimethylation of H3K4 by inhibiting the binding of an ASH2/WDR5/MLL-family methyltransferase complex that is responsible for establishing the H3K4me3 mark. Reciprocally, H3K4me3 prevents H3R2 dimethylation by the arginine methyltransferase PRMT6 (Guccione et al., 2007). While the so far discussed roles of H3K4me3 are clearly connected to gene activiation, it is surprising that other protein complexes involved in transcriptional repression like the Sin3-HDAC1 deacetylation complex or the JMJD2A demethylase can also be recruited by the H3K4me3 mark. A possible explanation would be that H3K4me3 establishes gene activation but afterwards reinstates a repressional state (Berger, 2007). This example shall illustrate how post-translational modifications are connected via crosstalk mechanisms, a regulatory principle that is realized not only for post- translational modifications of histones but also of other proteins (Hunter, 2007).

Figure 1. Post-translational modifications of core histones. This figure is adapted from a SnapShot on histone-modifying enzymes (Kouzarides, 2007b). Depicted are only those post-translational histone modifications, for which the associated enzymes were identified. Ac: acetylation; Me: methylation; P: phosphorylation; Ub: ubiquitination

Introduction 3

1.2 ADP-ribosylation: an unusual post-translational modification The modification of proteins by ADP-ribosylation is a phylogenetically ancient mechanism. It involves the transfer of ADP-ribose from NAD+ to target proteins accompanied by the release of nicotinamide. The consumption of NAD+ by ADP- ribosylating enzymes connects them not only to the energy metabolism but also to other NAD+-consuming enzymes including histone deacetylases of the Sirtuin family and ADP-ribosyl cyclases (Ying, 2006). NAD+, found in a cytosolic concentration of about 500 M and possessing a half-life of about 1-2 h (Elliott and Rechsteiner, 1975; Rechsteiner et al., 1976; Williams et al., 1985), and its reduced form NADH act as co- enzymes in numerous redox reactions, playing pivotal roles in many fundamental cellular processes, including mitochondrial oxidative phosphorylation as co-factors for three rate-limiting enzymes of the TCA cycle. In addition, NAD+ has been implicated in affecting cell death, in the regulation of calcium homeostasis and in controlling gene expression (Ying, 2006). Thus, the consumption of NAD+ by NAD+-consuming enzymes has to be tightly controlled.

The presence of poly-ADP-ribose (pADPr) was first described by P. Chambon and co- workers in 1963 (Chambon et al., 1963) and subsequently its structure was solved (Doly and Mandel, 1967; Reeder et al., 1967; Sugimura et al., 1967). The gene encoding the responsible enzyme named poly-ADP-ribose polymerase (PARP) was isolated in the late 1980s (Alkhatib et al., 1987; Kurosaki et al., 1987; Uchida et al., 1987). In the meantime another ADP-ribosylation reaction was discovered during the analysis of bacterial toxins (Gill et al., 1969; Honjo et al., 1968). While PARP enzymes catalyze the poly-ADP-ribosylation of proteins, the discovered toxins were shown to mono-ADP-ribosylate proteins. Consequently, the presence of mammalian mono- ADP-ribosyltransferases (mART) was suggested and ecto-enzymes with mART activity were discovered, while the molecular identity of postulated intracellular enzymes is less clear (Corda and Di Girolamo, 2003; Hassa et al., 2006; Okazaki and Moss, 1996; Okazaki and Moss, 1999; Seman et al., 2004). Recent evidence suggests that mitochondrial SIRT4 and nuclear SIRT6 possess mono-ADP-ribosyltransferase activity (Haigis et al., 2006; Liszt et al., 2005), potentially contributing to the mono-ADP- ribosylation of intracellular proteins.

1.3 The PARP superfamily of ADP-ribosylating enzymes PARP1, the founding and best-studied member of the PARP family, was for a long time considered to be the only enzyme in mammalian cells that could generate pADPr polymers. However, in recent years additional enzymes have been described and Introduction 4 systematic studies have indicated that 17 different proteins exist that share a catalytic PARP domain and thus may contain polymerase activity (Ame et al., 2004; Otto et al., 2005). Besides the PARP domain a wide spectrum of protein domains are present in the different members of the PARP family, suggesting that these proteins are involved in a broad spectrum of physiological activities (Figure 2).

Figure 2. The PARP superfamily (from (Schreiber et al., 2006)). The domain architecture of the 17 members of the poly(ADP-ribose) polymerase (PARP) superfamily and of poly(ADP-ribose) glycohydrolase (PARG). Protein-protein interaction motifs: BRCT (BRCA1 C-terminus), WWE (domain with conserved WWE motif), SAM (sterile -motif), ANK (ankyrin), vWA (von Willebrand factor type A), MVP-BD (major vault protein binding domain); Nucleic acid binding motifs: Zn finger, DBD (DNA binding domain), RRM (RNA recognition motif); ADP-ribose (and derivatives) binding motif: macro; Motifs of unknown function: WGR (Trp-Gly-Arg motif), HPS (homopolymeric His-Pro-Ser motif), VIT (vault inter--trypsin domain). Within each putative PARP domain, the region homologous to the PARP signature (residues 859–908 of PARP-1) is darkened. Please note that several members display splicing variants (e.g. PARP-3, PARP-9, PARP-14 and PARP-15), but for simplicity, only one variant is shown. MLS, mitochondrial localization signal; NES, nuclear export signal; NoLS, nucleolar localization signal; NLS, nuclear localization signal. Introduction 5

Indeed, the so far described members of the PARP superfamily display a significant diversity in their biological functions. The following paragraph will summarize the current state of knowledge about the analyzed PARP proteins.

1.3.1 PARP1 PARP1 represents the by far most intensively studied PARP enzyme and was described to participate in a wide range of cellular activities, including DNA repair, apoptosis and chromatin dynamics (D'Amours et al., 1999; Kim et al., 2005; Schreiber et al., 2006). Already in the late 1970’s, an intimate relationship between poly-ADP- ribosylation and a rapid and considerable decrease in cellular NAD+ levels after genotoxic stimuli was suggested (Goodwin et al., 1978; Skidmore et al., 1979). Indeed, in the presence of DNA strand breaks PARP1 activity and the levels of pADPr can be increased up to 500-fold with a concomitant reduction of NAD+ (Alvarez-Gonzalez and Althaus, 1989; Singh et al., 1985; Wielckens et al., 1983). The inhibition of PARP activity during DNA damage using chemical inhibitors can prevent the depletion of NAD+, ATP and deoxynucelotides (Das and Berger, 1986; Sims et al., 1983) suggesting a consumption of NAD+ by PARP1 during pADPr synthesis. The catalytic activation of PARP1 by DNA was shown to be dependent on the type of DNA, with undamaged DNA being ineffective, but with nicked DNA being a potent activator (Benjamin and Gill, 1980). This activation depends on a N-terminal DNA-binding domain in PARP1 containing two unusual zinc fingers (aa 21-56 and 125-162), with finger F1 being mainly responsible for activation by double-strand breaks (DSBs), while both fingers are necessary for the stimulation in response to single-strand breaks (SSBs) (Ikejima et al., 1990). The PARP1 zinc fingers are structurally and functionally unique and represent a DNA-break sensing motif. They coordinate zinc molecules with an unusual Cys-Cys-His-Cys motif and recognize altered DNA structures rather than particular sequences (Gradwohl et al., 1990). Interestingly, DNA can also serve as potent PARP1 activator in the absence of strands breaks. It was observed that PARP1 can be efficiently activated by binding to DNA hairpins, cruciform DNA and stable DNA loop regions (Lonskaya et al., 2005). A similar kind of zinc finger motif is found in the DNA repair enzyme DNA ligase III (Caldecott et al., 1996). Consistently, recent observations made by database searches suggest that PARP-like zinc fingers are evolutionary conserved in the eukaryotic lineage and associated with various enzymes implicated in nucleic acid transactions (Petrucco and Percudani, 2008). In addition to its zinc fingers, PARP1 contains additional DNA bindings motifs, which do not participate in enzymatic activation. Two helix-turn-helix motifs (aa 200-220 and 280- 285) (Saito et al., 1990; Uchida et al., 1987) mediate strong binding of PARP1 to Introduction 6 undamaged dsDNA and a fragment comprising these motifs behaves like the full-length enzyme in DNA footprinting assays (Buki and Kun, 1988; Sastry et al., 1989; Thibodeau et al., 1993).

Considering its DNA binding activities and its dramatic activation by DNA strand breaks, it is not surprising that PARP1 plays an important role in DNA repair processes. PARP1 is an integral component of the base excision repair pathway (BER), and its function can be explained by the ‘shuttling model’ (Figure 3): (1) SSBs are recognized by PARP1 resulting in full enzymatic activation; (2) activated PARP1 poly-ADP- ribosylates itself in an auto-modification reaction as well as histones H1 and H2B and possibly other proteins; (3) the poly-ADP-ribosylation of histones and/or of PARP1 itself causes relaxation of the chromatin and facilitates access to the site of the DNA lesion; (4) components of the BER pathway like XRCC1 and DNA ligase III are recruited by recognizing the locally produced pADPr; (5) poly-ADP-ribosylated PARP1 is repulsed from the DNA by electrostatic interference between the negatively charged DNA and the negatively charged pADPr; (6) the enzymatic activity of PARP1 is shut down to a basal level after the disruption of DNA binding; (7) the enzyme poly-ADP-ribose glycohydrolase efficiently removes the pADPr from PARP1 preparing it for another cycle of DNA damage recognition and initiation of DNA repair (Althaus et al., 1994; D'Amours et al., 1999; Schreiber et al., 2006; Zahradka and Ebisuzaki, 1982).

Figure 3. A model explaining PARP1’s role in DNA SSB repair (modified from(Schreiber et al., 2006)). For explaining text, see bottom of next page.

Introduction 7

Beside its role in the BER pathway, increasing evidence suggests that PARP1 is involved in an alternative DSB repair pathway. While DBSs are routinely repaired via the non-homologous end-joining (NHEJ) pathway involving the coordinated action of the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex, the BER components PARP1, XRCC1 and DNA ligase III are also capable to repair DSBs providing a backup pathway for the classical NHEJ mechanism (Audebert and Calsou, 2007; Audebert et al., 2004; Audebert et al., 2006). Interestingly, some components of the NHEJ pathway were shown to posses an pADPr binding motif mediating a non-covalent interaction with free pADPr and poly-ADP-ribosylated proteins (Althaus et al., 1999). Although PARP1 binds to DSBs and participates in their repair via an alternative non-homologous repair pathway, it is not involved in the repair of DSBs via homologous recombination (Helleday et al., 2005). However, it is clearly important for maintaining genomic stability (D'Amours et al., 1999; Herceg and Wang, 2001; Kim et al., 2005) and was shown to protect eroded telomeres via an interaction with the telomere repeat factor TRF2 (Gomez et al., 2006). Thus, it was proposed that the loss of PARP1 activity results in an increase of recombinogenic lesions, rather than PARP1 being involved in recombination as such (Helleday et al., 2005). This hypothesis is supported by two clinically important studies reporting the effectiveness of PARP1 inhibition in hereditary breast cancer (Bryant et al., 2005; Farmer et al., 2005). This familial form of breast cancer is in some cases caused by an inherited defect and subsequent loss of heterozygosity (LOH) in one of the BRCA1 or BRAC2 alleles, both playing important roles in the repair of DNA DSBs by homologous recombination (Venkitaraman, 2002). It was shown that BRCA deficient cells are hypersensitive to PARP1 inhibitors leading to massive cell death. Importantly, the inhibition of PARP1 in tumorigenic cells not only stopped the growth of the tumor in xenograft models, but resulted in a partially and sometimes complete remission (Bryant et al., 2005; Farmer et al., 2005).

Legend Fig. 3: PARP1 detects DNA lesions such as SSBs (red) resulting in DNA-dependent dimerization and subsequent enzymatic activation. The local synthesis of pADPr implements the relaxation of the chromatin around the site of the lesion and the attraction of DNA repair enzymes like XRCC1. Poly-ADP-ribosylated PARP1 is repulsed from the DNA resulting in disruption of the enzymatically functional homodimer and return of the enzymatic activity to basal levels. pADPr attached to PARP1 is rapidly degraded by PARG. The numbering refers to the text above that explains the ‘shuttling model’. The chromatin structure was taken from (Khorasanizadeh, 2004). Introduction 8

These observations led to the model (Figure 4) that spontaneously occurring SSBs cannot be repaired in the absence of PARP1 activity, in this case achieved by chemical inhibition of the enyzme, resulting in the collapse of replication forks. BRCA proficient cells are able to repair these profound lesions via homologous recombination, while in BRCA deficient cells these lesions are either repaired by error-prone pathways leading to further genetic instability or are directly lethal to cells (Helleday et al., 2005). Driven by these promising studies, several pharmaceutical companies have initiated oncology clinical trials with PARP1 inhibitors, and first promising results show the effectiveness of PARP1 inhibition in potentiating the activity of DNA-damaging agents (Ratnam and Low, 2007). But two very recent studies dampened the enthusiasm about the use of PARP1 inhibitors in cancer therapy, showing that BRCA2 deficient cells can evade the cell death mediated by PARP1 inhibitors through the acquisition of secondary mutations in the BRCA2 gene, which restore the protein function in homologous repair (Edwards et al., 2008; Sakai et al., 2008). Thus, tumor cells might get resistant to the chemotherapeutical agents by further mutations favorable to survive the cancer therapy, which will lead to recurrence of the tumor (Sakai et al., 2008).

Figure 4. Mechanism for specific killing of BRCA2 defective cells with inhibitors of PARP (from (Helleday et al., 2005)). PARP1 is involved in the efficient repair of spontaneous SSBs, lack of repair causes replication forks to collapse. Thus the number of collapsed replication forks is increased following treatment with PARP inhibitors. BRCA2 proficient cells efficiently repair collapsed replication forks with homologous recombination and resume replication without error. Cancer cells that lack functional BRCA2 fail to repair collapsed replication forks, these are either repaired by error-prone repair or are directly lethal to cells.

Introduction 9

PARP1’s role in DNA repair is closely linked to its role in the regulation of chromatin structure. Chromatin can be relaxed to the typical ‘beads on a string’ structure due to poly-ADP-ribosylation of histone H1 by PARP1, whereas the modified H1 remains associated with chromatin. Degradation of pADPr by PARG reverts the chromatin to a more condensed state (Aubin et al., 1983; de Murcia et al., 1986; Poirier et al., 1982). It was also shown that auto-modified PARP1 is able to dissociate chromatin independent of direct enzymatic activity via non-covalent interactions with histones which led to the proposal of a ‘histone shuttle’ mechanism: pADPr covalently attached to PARP1 could serve as a scaffold for sequestering histones producing a locally relaxed chromatin state. Subsequent cleavage of pADPr by PARG would then free the histones and allow histone-DNA complexes to rebuild (Althaus, 1992; Althaus et al., 1994; Realini and Althaus, 1992). Consistently, a study analyzing polytene chromosomes in Drosophila, which possesses only two PARP genes, i.e. a PARP1 and a PARP5 orthologue (Adams et al., 2000; Hanai et al., 1998), shows that the chemical inhibition of PARP activity as well as the blockage of PARP1 expression inhibits the synthesis of pADPr, chromatin decondensation, and the transcription of genes induced by heat shock or hormones (Tulin and Spradling, 2003). The same group also published at the first glance contradictory results showing that dPARP1 promotes the formation of compact chromosome structures in Drosophila (Tulin et al., 2002). Genetic disruption of PARP1 expression resulted in larval lethality, failure in nucleoli formation and in decondensation of heterochromatic, but not euchromatic regions. Interestingly, larval lethality could be restored by expression of a PARP1 mutant predicted to lack enzymatic activity, suggesting a role for PARP1 in the organization of heterochromatin independent of its catalytic activity (Tulin et al., 2002). Three studies from the Kraus lab support this idea and provide significant insight into PARP1’s function in heterochromatin formation (Kim et al., 2004; Krishnakumar et al., 2008; Wacker et al., 2007). The first study shows that PARP1 incorporates into chromatin by virtue of nucleosome-binding properties resulting in chromatin compaction and transcriptional repression, a function reminiscent of the linker histone H1. The binding to nucleosomes stimulates the auto-poly-ADP-ribosylation of PARP1 leading to the release of PARP1 from chromatin and subsequent chromatin relaxation, a finding contrary to the models described above which favor either a relaxation of chromatin by the poly-ADP- ribosylation of histones or a stripping of histones from the DNA caused by their interaction with poly-ADP-ribosylated PARP1 (Kim et al., 2004). Recent observations support PARP1’s role in chromatin compaction (Wacker et al., 2007). Using atomic force microscopy, the authors show that the DBD of PARP1 is necessary and sufficient for binding to nucleosomes, but is neither efficient in chromatin compaction nor Introduction 10 completely efficient in transcriptional repression. Instead, the catalytic domain of PARP1 cooperates with the DBD to promote the compaction of chromatin and transcriptional repression independent of its catalytic activity (Wacker et al., 2007). A very recent study using a genome-wide ChIP-chip approach suggests that PARP1 and histone H1 exhibit a reciprocal binding pattern at many RNA polymerase II-transcribed promoters, with PARP1 being enriched around the transcriptional start sites of actively transcribed genes (Krishnakumar et al., 2008). In addition, a subset of genes positively regulated by PARP1 showed a significant increase in H1 binding after PARP1 knockdown providing a functional link between PARP1 and H1 at these promoters (Krishnakumar et al., 2008). In summary, an important role of PARP1 in general chromatin compaction and in the transcriptional regulation of certain promoters seems likely, although Parp1-/- mice show no developmental abnormalities (Wang et al., 1995), which would be expected from a major regulator of chromatin structure.

Beside PARP1’s role in regulating transcription via the modulation of chromatin structure, several studies indicate that it can work as a specific cofactor for certain transcription factors (reviewed in (Hassa and Hottiger, 2008; Kim et al., 2005; Schreiber et al., 2006)). Expression profiling in Parp1-/- mouse embryonic fibroblasts (MEFs) revealed that about 1% of 11,000 analyzed genes were differentially expressed in comparison to MEFs derived from wild-type littermates, with Parp1 loss resulting in the down-regulation of several genes involved in DNA/RNA repair or synthesis and up- regulation of many genes encoding extracellular matrix or cytoskeletal proteins (Simbulan-Rosenthal et al., 2000). A further study analyzed the expression of inflammatory cytokines and NF-B-dependent genes in LPS-stimulated glia cells of Parp1+/+ and Parp1-/- mice observing a great reduction in IL-6, IL-1ß, ICAM-1, and vesicular adhesion molecule-1, while the expression of the majority of analyzed genes was not changed significantly (Ha et al., 2002). However, Western Blot analysis of protein expression after several time points following LPS-stimulation showed a broader role of PARP1 in the LPS-induced activation of cytokine expression, with catalytic activity being relevant for the expression of only some PARP1-dependent genes (Ha et al., 2002). A recent expression profiling in Parp1-/- ES cells and liver cells shows that almost 10% of the genes in ES cells show altered expression in the Parp1-/- background and 3.3% in the liver cells, with a majority of the genes being down- regulated in the absence of Parp1 (Ogino et al., 2007). Consistent with these expression studies, PARP1 was suggested to act as transcriptional co-factor for NF- B, E2F-1, B-MYB, AP-2, PAX6, TEF1 and the viral protein Tax, a function that is not necessarily dependent on its ADP-ribosylation capacity (Anderson et al., 2000; Butler Introduction 11 and Ordahl, 1999; Cervellera and Sala, 2000; Hassa et al., 2003; Kannan et al., 1999; Plaza et al., 1999; Simbulan-Rosenthal et al., 2003). Other studies implicate a role for PARP1 in assisting transcriptional repression, e.g. in repressing its own expression (Soldatenkov et al., 2002) or as a co-repressor of the nuclear hormone receptor RXR (Miyamoto et al., 1999). Again a general dependency on its catalytic activity could not be shown for the co-repressor function and seems to depend on the interacting transcription factor.

Besides its function as classical co-activator or co-repressor in transcription, some studies provide evidence for a role of PARP1 as a co-repressor/co-activator exchange factor at certain promoters (Hassa et al., 2005; Ju et al., 2006; Ju et al., 2004; Pavri et al., 2005). In the context of NF-B-dependent gene activation, PARP1 is able to physically interact with the co-activator complex Mediator and the histone acetyl transferase p300 as well as with the histone deacetylases HDAC1-3 (Hassa et al., 2005). Acetylation of several lysine residues in PARP1 by p300/CBP results in the enhanced interaction with p50 and subsequent activation of NF-B-dependent gene expression by p300 and the Mediator complex in response to inflammatory stimuli, suggesting a stimulus-dependent induction of PARP1-dependent NF-B activation in concert with further transcriptional co-activators (Hassa et al., 2005). An interplay between PARP1 and Mediator was also observed when studying the essential factors for retinoic acid receptor (RAR)-mediated transcription from the RARß2 promoter (Pavri et al., 2005). PARP1 occupies RAR-responsive promoters continuously along with the Mediator complex and other factors as confirmed by ChIP experiments, but after ligand-dependent induction of gene expression it is indispensable for converting Mediator from its inactive state (Cdk8+) to its active state (Cdk8-) through the release of the repressing Cdk8 module (Pavri et al., 2005). Interestingly, PARP1 directly binds Cdk8 via its N-terminal region, and the interaction is weakened after TNF- stimulation, which precedes the acetylation of PARP1 and subsequent NF-B activation (Hassa et al., 2005). A similar situation was observed in neural differentiating stem cells: PARP1 as component of the groucho/TLE co-repressor complex mediates the release of this complex upon PDGF stimulation and, after PDGF-triggered activation of CaMKII, allows for the recruitment of co-activators like CBP and subsequent promoter activation (Ju et al., 2004). The catalytic activity is essential for this function and several components of the groucho/TLE complex including TLE1, the DNA topoisomerase TopoIIß and Nucleolin get ADP-ribosylated by PARP1 (Ju et al., 2004). The authors extended their model by a further study providing evidence that the signal-dependent activation of transcription requires the transient formation of dsDNA breaks by TopoIIß Introduction 12 and concomitant PARP1 activation, the latter inducing a nucleosome-specific exchange of histone H1 against the high-mobility group (HMG) B protein and local changes in chromatin structure (Ju et al., 2006).

Another physiological role of PARP1 is its closely linked involvement in inflammation and apoptosis (Figure 5). As mentioned above, PARP1 triggers the expression of pro- inflammatory genes after exposure to external stress signals like ischemia or LPS resulting in the generation of reactive oxygen species, PARP1 activation, translocation of AIF to the nucleus, DNA degradation, subsequent excess PARP1 activation with concomitant NAD+ and ATP depletion, and finally caspase-independent, necrotic cell death which promotes an inflammatory response in the surrounding tissue (Schreiber et al., 2006). An alternative apoptotic route, which is taken in the presence of limited DNA damage, is p53-dependent and involves the release of cytochrome c and activation of the caspase cascade (Schreiber et al., 2006).

Figure 5. PARP at the crossroad of survival and inflammation (from (Schreiber et al., 2006)). For explaining text, see bottom of next page.

Introduction 13

In agreement with this proposed action of PARP1 are the data obtained from studies on knockout mice. In various models of inflammation, including diabetes and septic shock, Parp1-/- mice show enhanced resistance (Hassa and Hottiger, 2002; Mabley et al., 2001; Oliver et al., 1999).

In summary, PARP1 is involved in a wide variety of physiological processes with its catalytic activity being important for some, but not all of its functions. How regulation of PARP1’s role in these processes is achieved is poorly understood and has to be elucidated in the future.

Legend for Fig. 5: In response to inflammatory stress, NF-B–PARP1 induce the transcription of pro- inflammatory genes (e.g. the inducible nitric oxide synthase (iNOS) gene) in macrophages. The subsequent production of nitric oxide (NO) and reactive oxygen species (step 1) triggers DNA- strand breaks (for example, in postmitotic cells) (step 2) that activate PARP1 and PARP2 (step 3). Poly(ADP-ribose) (PAR) has a short half-life and is rapidly degraded by PARG. This reaction produces an as yet unidentified molecular signal, which is possibly free PAR, that is transmitted from the nucleus to the mitochondria (step 4), where it induces a reduction of the mitochondrial membrane potential (m) and the release of apoptosis-inducing factor (AIF) and possibly endonuclease G (EndoG) (step 5). AIF translocates into the nucleus where it induces the degradation of DNA into high-molecular weight fragments (chromatinolysis, step 6). This overactivates PARP1 and PARP2 in a second wave of strong PAR synthesis (step 7) that leads to NAD+ and ATP depletion (step 8) and, finally, cell death (step 9). In proliferating cells that face a limited amount of DNA damage, PARP inhibition impairs DNA repair and promotes cell death via apoptosis, mainly through p53 activation. p53 modifies the expression of anti-apoptotic (BCL2) and pro-apoptotic (BAK) factors that regulate the release of cytochrome c (cyto c), which subsequently associates with Apaf1 (not shown) and procaspase- 9. This triggers the caspase-activation cascade, the activation of inhibitor of caspase-activated DNAse (iCAD)–CAD, which promotes DNA fragmentation, and the cleavage of PARP1 to avoid futile DNA repair. IB, inhibitor of NF-B; IL, interleukin; Pol II, RNA polymerase II; TNF, tumor-necrosis factor. Introduction 14

1.3.2 PARP2 PARP2 was discovered in the late 1990s and displays high homology to the C-terminus of PARP1 harboring the catalytic domain and also contains a N-terminal DNA-binding domain (Ame et al., 1999). As expected, its catalytic properties strongly resemble those of PARP1, with the synthesis of long pADPr polymers being dependent on the binding to and activation by nicked DNA (Ame et al., 1999). Consequently, it was observed that PARP2 shares many functions with PARP1 in DNA repair processes: it interacts with the same components of the BER pathway, its enzymatic activity is required for an efficient BER of DNA lesions, it is able to hetero-dimerize with PARP1 within the same regions being responsible for its homo-dimerization, and PARP1 and PARP2 poly- ADP-ribosylate each other in a trans-ADP-ribosylation reaction (Schreiber et al., 2002). In addition, Parp2-/- mice are hypersensitive to DNA-damaging agents similar to Parp1-/- mice, and the double mutant Parp1-/- Parp2-/- mice are embryonically lethal demonstrating an essential role in early embryogenesis and underlining their at least partial functional redundancy in maintaining genome integrity (Menissier de Murcia et al., 2003). Further similarities between PARP2 and PARP1 include their interaction with TRF2 and a role in telomere protection (Dantzer et al., 2004; Gomez et al., 2006), the cleavage of PARP2 and PARP1 during caspase-dependent apoptosis, mediated by caspase-8 or caspase-3, respectively (Benchoua et al., 2002; Kaufmann et al., 1993; Tewari et al., 1995), and their localization to centromeres where they interact with and poly-ADP-ribosylate centromeric proteins (Saxena et al., 2002).

1.3.3 PARP3 PARP3 can undergo auto-ADP-ribosylation as described for PARP1 and PARP2 and was found to be associated with the centrosome where it preferentially interacts with the daughter centriole throughout the cell cycle (Augustin et al., 2003). PARP3 overexpression interferes with the G1/S phase cell cycle progression, and it was described to interact with PARP1 at centrosomes (Augustin et al., 2003). A recent study disputed the centrosomal localization of PARP3, but suggested a preferentially nuclear localization, an association with polycomb group (PcG) bodies and an interaction with PcG protein complexes (Rouleau et al., 2007). In addition, PARP3 was identified as part of DNA repair protein complexes by a proteomic approach, revealing an association e.g. with PARP1 and the DNA ligases III and IV (Rouleau et al., 2007). Thus, PARP3 might be involved in the maintenance of transcriptional repression and might be linked to the DNA repair machinery.

Introduction 15

1.3.4 PARP4 PARP4 (vPARP) was originally identified as a component of mammalian ribonucleoprotein complexes called vaults (Kickhoefer et al., 1999). It interacts with and ADP-ribosylates the major vault protein, undergoes auto-ADP-ribosylation and portions of the cellular PARP4 co-localize with cytoplasmic vault particles, while in mitotic cells PARP4 shows co-localization with the mitotic spindle (Kickhoefer et al., 1999). The incorporation of PARP4 into vault-like MVP-containing particles depends only on the C- terminal domain lacking catalytic activity, and the incorporation into vault complexes leads to the stabilization of PARP4 and MVP (Zheng et al., 2005). Another study shows an interaction of PARP4 with the telomerase-associated protein 1 (TEP1), but the analysis of Parp4-/- mice revealed that these mice are viable and fertile without any apparent defects in telomer length or function and in the structure of vault particles (Liu et al., 2004). Thus, PARP4 is dispensable for the function of vaults as wells as for maintaining telomere function. The only phenotype observed so far for Parp4-/- mice is an increased susceptibility to carcinogen-induced colon tumorigenesis (Raval- Fernandes et al., 2005).

1.3.5 PARP5a and PARP5b (Tankyrase1 and 2) PARP5a and 5b are two closely related members of the PARP superfamily whose functions were mainly connected to telomeres, although additional roles in mitosis and vesicular trafficking have been proposed (reviewed in (Hsiao and Smith, 2008)). PARP5a (Tankyrase1) was identified in a yeast-two hybrid screen as an interaction partner for the telomeric repeat binding factor 1 (TRF1), with binding to TRF1 being mediated through the ankyrin repeats of PARP5a (Smith et al., 1998). It is able to catalyze auto-poly-ADP-ribosylation, poly-ADP-ribosylates TRF1 and co-localizes with TRF1 at telomeric DNA (Smith et al., 1998). A careful analysis of its ADP-ribosylation capacity revealed that PARP5a is able to synthesize pADPr with an average chain length of 20 units but lacks the ability to form branched polymers as described for PARP1 and PARP2 (Rippmann et al., 2002). The ability of PARP5a to ADP-ribosylate TRF1 depends on TRF1 binding through the ankyrin repeat cluster (ARC) V (Seimiya et al., 2004). PARP5a’s ability to associate with telomeres was shown to be dependent on TRF1, and in addition to its telomeric localization it also resides at nuclear pore complexes during interphase and localizes to mitotic centrosomes (Smith and de Lange, 1999). Consequently, a role for PARP5a in the telomere elongation was proposed, suggesting that the ADP-ribosylation of TRF1 by PARP5a releases TRF1 from telomeres allowing access of telomerase to telomeres (Cook et al., 2002; Smith and de Lange, 2000). This release might be regulated by a second TRF1-interacting Introduction 16 factor, TIN2 (Ye and de Lange, 2004). In contrast to this model, a mutant PARP5a lacking ARC V and unable to ADP-ribosylate TRF1 seems to be sufficient to loosen the telomeric heterochromatin and to promote telomere elongation (Muramatsu et al., 2007b).

Beside its role in telomere function, PARP5a was described to be important for the cell to pass through mitosis correctly, a function being strictly dependent on its catalytic activity (Chang et al., 2005a; Dynek and Smith, 2004). Specifically, the knockdown of PARP5a results in mitotic arrest due to a failure in chromosomal segregation. While one study connected this observation to a post-anaphase arrest induced by failure of sister chromatid segregation at their telomeres (Dynek and Smith, 2004), the authors of another study suggest that PARP5a is required for the assembly of bipolar spindles with PARP5a knockdown resulting in a pre-anaphase arrest (Chang et al., 2005a). The proposed role in spindle formation is supported by the fact that the essential spindle protein NuMA was found to be a substrate for PARP5a-mediated poly-ADP-ribosylation in mitosis (Chang et al., 2005a; Chang et al., 2005b). The knockdown of PARP5a resulted in the loss of poly-ADP-ribosylated NuMA, but NuMA was still localized correctly to the spindle poles. In contrast, knockdown of NuMA completely abolished the localization of PARP5a to spindles during mitosis, indicating an essential role for NuMA in the recruitment of PARP5a (Chang et al., 2005b). A potential regulation of PARP5a in mitosis might be achieved through the phosphorylation by GSK3 (Yeh et al., 2006).

While most of the PARP5a molecules are associated with the nuclear envelope during interphase (Smith and de Lange, 1999), a cytoplasmic portion was shown to be associated with the Golgi (Chi and Lodish, 2000). Closer analysis revealed an interaction of PARP5a with the insulin-responsive amino peptidase (IRAP) that likely recruits it to GLUT4 storage vesicles in adipocytes (Chi and Lodish, 2000).

An interesting aspect of crosstalk between different members of the PARP superfamily was reported for PARP5a and PARP1 (Yeh et al., 2005). While the overexpression of PARP5a in MDCK cells resulted in lower steady-state NAD+ levels without affecting cellular viability, it rendered the cells more resistant to the cytotoxic effects mediated by

H2O2 and the DNA-alkylating agent MNNG. This effect was due to the inhibition of PARP1 activation and concomitant NAD+ consumption, thus providing evidence for a cytoprotective function of PARP5a mediated through crosstalk with PARP1 (Yeh et al., 2005). Introduction 17

The gene encoding PARP5b, at that time designated as the TNKL gene, was cloned in 2001 and located to chromosome 10q23-24 (Kuimov et al., 2001). Almost simultaneously, the PARP5b protein was identified as an interaction partner of the adaptor protein Grb14, and both proteins were shown to be enriched in a subcellular fraction containing Golgi vesicles and endosomes (Lyons et al., 2001). At the same time, an association of PARP5b with TRF1 was reported, and strong overexpression of PAPR5b was found to cause cell death dependent on its catalytic activity (Kaminker et al., 2001). The findings of both reports remind of the ones obtained for PARP5a (see above). Consistently, the poly-ADP-ribosylation of TRF1 by PARP5b and the subsequent release of TRF1 from telomeres was described, indicating a potentially redundant role for PARP5a and PARP5b in telomere regulation (Cook et al., 2002). Considering the high homology of both tankyrases (see Figure 2), these observations were not surprising. Indeed, the discovery of a tankyrase-binding motif in several of the interaction partners shared between both tankyrases (Sbodio and Chi, 2002) and their reciprocal ability to associate and to colocalize (Sbodio et al., 2002) suggest a high redundancy in terms of physiological function, a situation resembling the close connection between PARP1 and PARP2 (see above). Interestingly, the analysis of Parp5b-/- mice revealed a significant decrease in the body weight pronounced in male mice, but no differences in telomere length as expected from the proposed role in the maintenance of telomere length (Chiang et al., 2006). Studies on transgenic mice carrying a Parp5b gene with a deletion of the catalytic domain presented similar results, showing no effects on telomere length and telomere capping, but a growth retardation phenotype (Hsiao et al., 2006). An explanation for the normal telomeres in these mice might be that murine TRF1 does not contain a tankyrase-binding motif as its human counterpart and consequently does neither interact with PARP5a nor does it get modified by it (Muramatsu et al., 2007a). Due to the high telomerase activity in rodents compared to mammals and their long telomeres, these species might have evolved to get along without the tankyrase-mediated maintenance of telomere length (Muramatsu et al., 2007a).

1.3.6 PARP7 (TiPARP) Parp7 was identified as a gene upregulated by the treatment of mouse hepatoma cells with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (Ma et al., 2001), a halogenated aromatic by-product of industrial processes causing pleiotropic harmful effects in mammalian species because it serves as ligand for the cytoplasmic receptor AhR and Introduction 18 induces its translocation to the nucleus where it hetero-dimerizes with its partner protein Arnt and binds to enhancer sequences called dioxin-response elements (DREs) (Ma, 2001). Parp7 is broadly expressed in murine tissues and likely possesses enzymatic ADP-ribosylation capacity, although it was not determined whether it undergoes auto-ADP-ribosylation (Ma et al., 2001). Since a partial sequence of the rat Rm1 gene is homologous to the 3’ end of Parp7, it was speculated that Parp7 might be the murine homologue of the rat protein RM1 that is proposed to play a role in memory formation (Matsuo et al., 2000), even though no experimental data exist for this hypothesis (Ma et al., 2001).

1.3.7 PARP9, 14, 15 (BAL1, 2, 3 / macroPARPs) The gene encoding PARP9 was originally identified as BAL1 (B-aggressive lymphoma 1) gene in a screen for risk-related genes in diffuse large B cell lymphoma (DLBCL) and mapped to chromosome 3q21 (Aguiar et al., 2000). It encodes a nuclear protein of 88 kDa with a N-terminal duplicated macro domain (Aguiar et al., 2000), a protein motif involved in the detection of different forms of ADP-ribose and in transcriptional repression (discussed below). The overexpression of PARP9 in B-cell lymphoma cells stimulated their migration in transwell assays, indicating a tumor-promoting role for PARP9 in malignant B cells (Aguiar et al., 2000). Interestingly, PARP9 interacts with the E3 ligase BBAP (Takeyama et al., 2003), and the expression of both genes is regulated by an interferon--responsive bidirectional promoter (Juszczynski et al., 2006). BBAP regulates the subcellular localization of PARP9, and PARP9 overexpression in DLBCL cell lines resulted in an increase in the expression of multiple interferon-stimulated genes, suggesting an important role for PARP9 in inflammatory processes (Juszczynski et al., 2006). Despite the fact that PARP9 contains a C- terminal PARP domain, it lacks catalytic activity due to the absence of conserved residues essential for catalysis (discussed below), but it is able to repress transcription in general via its macro domains as assessed by reporter gene assays (Aguiar et al., 2005).

A database search identified two genes related to BAL1, which consequently were named BAL2 and BAL3 (Aguiar et al., 2005). Both genes were mapped to chromosome 3q21 arranged in tandem with the previously identified BAL1 (Aguiar et al., 2005). The encoded proteins, PARP14 and PARP15, like PARP9 possess N-terminal macro domains and a C-terminal PARP domain, but in contrast to PARP9, the C-terminal region of both proteins is subject to an auto-ADP-ribosylation indicating an intact catalytic activity (Aguiar et al., 2005). PARP15 was reported to have a transcriptionally Introduction 19 repressive function, which is not as pronounced as for PARP9 but also mediated by its macro domains. Since its PARP domain alone slightly stimulated transcription, it was suggested that the intrinsic PARP activity of PARP15 might counteract the repressive effect of the macro domains (Aguiar et al., 2005).

PARP14 was identified in a yeast two-hybrid screen as interaction partner of STAT6 and was named collaborator of STAT6 (CoaSt6) (Goenka and Boothby, 2006). In spite of its three macro domains and in contrast to the repressive effect of the related proteins PARP9 and PARP15, PARP14 was described to have a potentiating effect on the IL-4-induced transcriptional activation by STAT6 mediated mainly by its macro domains (Goenka and Boothby, 2006). Importantly, PARP14 seems to be a specific coactivator for the STAT6-mediated transcription, since it failed to stimulate the interferon--induced gene expression by STAT1 (Goenka and Boothby, 2006). Although the macro domains were described to be sufficient for the co-activator function of PARP14 in reporter gene assays as well as for the endogenous IL-4- induced expression of the STAT6 target CD23 (Goenka and Boothby, 2006), the same authors suggest in their more recent study that the intrinsic PARP activity of PARP14 is essential for its STAT6 co-activator function (Goenka et al., 2007). They propose a model in which PARP14 ADP-ribosylates p100, a STAT6 co-activator enhancing the interaction of STAT6 with the basal transcription machinery, thereby mediating a stimulatory effect on the STAT6-driven gene expression (Goenka et al., 2007). The obvious discrepancy between these two studies from the same group is hard to explain at the current state of knowledge and will require further investigations. Referring to the physiological role of PARP14, it is interesting to note that in a subset of DLBCLs a high expression of components of the IL-4 signaling pathway is observed (Guiter et al., 2004; Lu et al., 2005), whereas other subsets of DLBCLs exhibit changes in the interferon- signaling pathway (Juszczynski et al., 2006). It is tempting to speculate that PARP14 and PARP9 might have antagonistic functions in DLCBLs, with one involved in IL-4 and the other involved in interferon- signaling.

1.3.8 PARP13 (ZAP(L)) The rat orthologue of PARP13 was identified by its ability to significantly impair the replication of murine leukaemia virus and lacks a PARP domain (Gao et al., 2002). Its antiviral property was linked to the specific binding and degradation of viral mRNAs in the cytoplasm (Gao et al., 2002), and the binding was shown to be mediated by the four CCCH zinc finger motifs in ZAP (Guo et al., 2004). The degradation of viral mRNAs involves the recruitment of the RNA processing exosome through an Introduction 20 interaction of ZAP and the exosome component hRrp46p (Guo et al., 2007). Interestingly, expression of the murine ZAP gene is induced by interferon- and -ß in a PKR-independent fashion, and its protein product inhibits Sindbis virus replication, thereby linking the ZAP protein to the interferon-mediated antiviral activities (Zhang et al., 2007). Interestingly, the human ZAP gene can be expressed in two alternatively spliced isoforms (Kerns et al., 2008). While the short isoform ZAP(S) does not code for a PARP domain, the longer isoform ZAP(L) encodes a protein containing a C-terminal PARP domain in addition to the CCCH zinc finger motifs, the TiPARP homology domain and a WWE domain (Kerns et al., 2008). The longer isoform was also named PARP13 (Ame et al., 2004). Both isoforms are expressed in a broad range of tissues, with ZAP(S) showing in general significantly higher expression levels (Kerns et al., 2008). Careful analysis of the rat gene also revealed the presence of a longer ZAP isoform containing a PARP domain. Since no ESTs have been reported for this PARP domain, it is likely that rat ZAP(L) is expressed at lower levels than ZAP(S) (Kerns et al., 2008). Evolutionary analysis provides strong evidence for a positive selection of the PARP domain within ZAP(L) throughout primate evolution, and the PARP domain increased the antiviral properties of ZAP against both retroviruses and alphaviruses (Kerns et al., 2008). Thus, although it is unclear whether the PARP domain in ZAP(L) is functional in terms of catalysis, it is likely that its physiological role is connected to antiviral effects.

1.3.9 PARP10 PARP10 exhibits a unique domain composition among the PARP family members, combining potential DNA/RNA binding motifs and ubiquitin interacting motifs with the catalytic PARP domain (Figure 6).

Figure 6. The domain architecture of PARP10. Indicated are the different domains present in PARP10 as derived from database prediction of conserved protein motifs. The numbering refers to the PARP10 amino acids. In the C-terminal region the catalytic core domain and the residues involved in binding of c-MYC are marked. RRM, RNA recognition motif; Gly, Glycine-rich region; NES, nuclear export sequence; Glu, Glutamate-rich region; UIM, ubiquitin interacting motif; PARP, catalytic PARP domain

Introduction 21

PARP10 was identified as an interaction partner of the prototypical oncoprotein c-MYC by affinity purification of a c-MYC protein complex from Jurkat T cells (Yu et al., 2005b). The PARP10 protein with an apparent molecular weight of 150 kDa was described as a catalytically competent PARP enzyme that ADP-ribosylates itself as well as core histones, but neither its interaction partner c-MYC nor the bHLH protein MAX, the heterodimerization partner of MYC (Yu et al., 2005b). The binding of PARP10 to c- MYC is mediated by a C-terminal region encompassing aa 700-907, and c-MYC binds PARP10 via its N-terminal region in a Myc box II (MBII)-dependent manner and via its C-terminal 176 amino acids (Yu et al., 2005b). Interestingly, both regions have been implicated in c-MYC’s ability to transform cells (Henriksson and Luscher, 1996).

The PARP10 gene was mapped by FISH analysis to chromosome 8q24 (Yu et al., 2005a), a region where also the c-MYC gene is localized (Henriksson and Luscher, 1996). The analysis of chromosomal translocation in Burkitt’s lympohoma cell lines revealed a localization of PARP10 telomeric from c-MYC, which afterwards could be specified to 8q24.3 (Yu et al., 2005b). A closer inspection of the chromosomal Parp10 locus in mouse exhibited an overlap of the most 3’ exons 10 and 11 of Parp10 with the non-coding exons -1 and 0a of the plectin1 gene. In addition, a hypothetical plectin1 promoter localized in intron 9 of Parp10 was suggested (Lesniewicz et al., 2005). This head-to-tail arrangement is likely conserved in rat and human and might potentially account for intergenic regulation of gene expression (Lesniewicz et al., 2005). Orthologues of PARP10 could be identified in rodents and in Fugu, but not in invertebrates suggesting that PARP10 has evolved in the vertebrate lineage (Lesniewicz et al., 2005). A single 3.8 kb mRNA transcript could be detected in a broad range of human tissues suggesting a ubiquitous expression with some preference for hematopoietic tissues (Yu et al., 2005b).

PARP10 was reported to be localized preferentially in the cytoplasm and to shuttle between the cytoplasm and the nucleus in a Crm1-dependent fashion dependent on its functional NES (Yu et al., 2005b). Importantly, PARP10 was able to impair the c- MYC/Ha-RAS-driven transformation of rat embryo fibroblast in a dose-dependent manner which is abrogated in a PARP10 mutant carrying a non-functional NES. Additionally, PARP10 inhibited the E1A/Ha-RAS-driven transformation suggesting that PARP10 might serve as a potent inhibitor of transformation in general. In both cases its catalytic properties seems dispensable for its function (Yu et al., 2005b). The analysis of starved 3T3-L1 cells re-entering the cell cycle after stimulation with FCS suggest that Introduction 22 overexpression of PARP10 in these cells interferes with progression into S-phase (Yu et al., 2005b).

Another study shows that PARP10 gets phosphorylated at Threonine-101 by Cyclin E/CDK2, and its phosphorylated form is localized to the nucleolus during interphase and to the RNA-Pol I containing nucleolus organizing regions during mitosis (Chou et al., 2006). The authors observed an accumulation of HeLa cells in G1-phase after knockdown of PARP10 by shRNA accompanied by a decrease in cell number suggesting an important role for PARP10 in cell viability (Chou et al., 2006). In addition, it was shown that the phosphorylated form of PARP10 is preferentially present in proliferating cells, which led the authors to speculate that their antibody recognizing the phosphorylated PARP10 might be useful as a biomarker for proliferation (Chou et al., 2006).

1.4 ADP-ribose binding motifs The first indication for an ADP-ribose binding motif was published in the late 1990s: three regions within the tumor suppressor protein p53, two in its core DNA binding domain and one in its oligomerization domain, were observed to bind pADPr, either free or attached onto PARP1, through strong non-covalent interactions (Malanga et al., 1998). The binding of p53 to pADPr interfered with its DNA-binding properties in gel shift assays, suggesting a potential regulation of p53’s functions by pADPr (Malanga et al., 1998). Interestingly, p53 was also found to undergo covalent poly-ADP-ribosylation by PARP1 which impairs its DNA binding properties (Mendoza-Alvarez and Alvarez- Gonzalez, 2001) and was suggested to block the Crm1-dependent export of p53, thereby resulting in nuclear p53 accumulation in the presence of DNA damage (Kanai et al., 2007). Further studies led to the discovery of a pADPr-binding motif composed of a cluster rich in basic amino acids and a pattern of hydrophobic amino acids interspersed with basic residues (Pleschke et al., 2000). This motif is not only present in p53, but also in several DNA repair enzymes like DNA ligase III or XRCC1, in NF-B, iNOS and telomerase (Pleschke et al., 2000). Surprisingly, all of these proteins were previously reported either as interaction partner/substrate of PARP1 or to act in the same functional pathway as PARP1 (discussed above). Thus, the non-covalent recognition of pADPr might add an additional layer of complexity to the PARP1- dependent regulation of these proteins.

Recently, it was described that the macro domain serves as high-affinity ADP-ribose binding module (Karras et al., 2005). Using ITC binding assays, the authors reported Introduction 23 specific binding of the macro domain from Af1521, a protein from the thermophilic organism Archaeoglobus fulgidus, to ADP-ribose with a KD in the range of 120 nM (Karras et al., 2005). The binding to ADP was substantially diminished compared to ADP-ribose suggesting an important role for the distal ADP-ribose in recognition by the macro domain. This is supported by the crystal structure of Af1521 with bound ADP- ribose showing that the ADP-ribose is bound in a L-shaped cleft on the protein surface with the adenine moiety residing in a deep hydrophobic pocket. Consistent with data from the binding assays, the distal ribose is able to form several H-bonds that are not present in the ADP-bound structure of Af1521, thus explaining the higher affinity for ADP-ribose (Karras et al., 2005). Importantly, the macro domains of additional proteins including the histone variant mH2A and PARP9 were also able to bind ADP-ribose. Furthermore it was observed that the macro domains of Af1521 and PARP9 are not restricted to recognize ADP-ribose, but are capable of binding pADPr (Karras et al., 2005). This finding was confirmed by analysis of viral macro domains that are able to bind ADP-ribose, free pADPr and PARP1-bound pADPr efficiently (Egloff et al., 2006). Unexpectedly, the macro domain of mH2A1.1 was not capable of recognizing pADPr, instead it binds 2’- or 3’-OAADPR, a metabolite produced during the deactylation reaction catalyzed by Sirtuins, with similar affinity as ADP-ribose (Kustatscher et al., 2005). Analyzing the splice variant mH2A1.2, the authors could show that subtle differences between the variants mH2A1.1 and mH2A1.2 in the ligand-binding pocket abrogate the binding of mH2A1.2 to OAADPR (Kustatscher et al., 2005).

Interestingly, the macro histone variants mH2A1 and mH2A2 are associated with the inactive X chromosome (Chadwick and Willard, 2001; Costanzi and Pehrson, 1998; Costanzi and Pehrson, 2001), and mH2A1 is not only concentrated on the inactive X chromosome but also depleted on active genes (Changolkar and Pehrson, 2006). This suggests a potential role for this histone variant in transcriptional repression. Indeed, mH2A1.2 was identified as a strong transcriptional repressor blocking transcription at the initiation step (Doyen et al., 2006). The repression is mediated by the non-histone region of mH2A1.2, the macro domain, and involves the repression of histone acetylation and nucleosome remodeling (Doyen et al., 2006). Since mH2A1.1 exhibits a different expression pattern in tissues and development compared to mH2A1.2 (Pehrson et al., 1997), further experiments are necessary to discriminate between the physiological roles of these highly related histone variants.

Two recent reports suggest an inhibitory role of mH2A1 towards the catalytic activity of PARP1 (Nusinow et al., 2007; Ouararhni et al., 2006), thereby linking the macro Introduction 24 domain as proposed ADP-ribose binding motif to the regulation of an ADP-ribosylating enzyme. Nusinow et al. observed an interaction between mH2A1.2 and PARP1’s zinc finger region and its catalytic domain, and the catalytic activity of PARP1 could be repressed by the macro domains of all mH2A family members (Nusinow et al., 2007). Importantly, the knockdown of PARP1 expression resulted in reactivation of a reporter gene embedded in the inactive X chromosome, which led to the proposal that mH2A might contribute to gene silencing by inhibiting PARP1 activity (Nusinow et al., 2007). In the study by Ouararhni et al., a direct interaction between PARP1 and mH2A1.1 was demonstrated and PARP1 could be identified as a component of mH2A1.1- nucleosomes (Ouararhni et al., 2006). Catalytic PARP1 activity of this nucleosomal complex was inhibited, while nucleosomes containing a mH2A1.1 mutant deficient in ADP-ribose binding showed normal PARP1 activity. Under stringent conditions PARP1 was released from these nucleosomes containing mutant mH2A1.1 but not from the mH2A WT nucleosomes, suggesting that the capacity to bind ADP-ribose mediates the recruitment and the catalytic inhibition of PARP1 by mH2A1.1 (Ouararhni et al., 2006). Importantly, the promoters of the inducible heat shock genes Hsp70.1 and Hsp70.2 were enriched in mH2A1.1 and bound PARP1, and heat shock released both proteins from the promoter dependent on PARP1’s catalytic activity thereby allowing transcriptional activation (Ouararhni et al., 2006).

In summary, the macro domain represents an ADP-ribose binding motif with distinct domains possessing slightly different specificities for ADP-ribose-related ligands and has been implicated in transcriptional repression. Further experiments need to clarify whether the macro domain functions as ADP-ribose binding motif in vivo, e.g. recognizing locally produced pADPr. The fact that mH2A1.1 is able to bind the metabolite OAADPR produced by enzymatically active Sirtuins, that all mH2A variants likely participate in transcriptional silencing and that mH2A1 potentially inhibits PARP1 activity in vivo suggests a functional interplay of Sirtuins, PARPs and the mH2A histone variants in regulating chromatin structure and gene expression. Future work will be needed to shed light on these functional network centered around ADP-ribose.

Recently, a third ADP-ribose binding motif termed pADPr-binding zinc finger (PBZ) was discovered (Ahel et al., 2008). It was reported that this C2H2 zinc finger is present in eukaryotic DNA repair and checkpoint control proteins and binds pADPr (Ahel et al., 2008). The binding of the PBZ-containing proteins CHFR and APLF to pADPr was significantly stronger than for the pADPr-binding protein XRCC1 and was abrogated by mutations targeting the PBZ consensus sequence (Ahel et al., 2008). Interestingly, Introduction 25

PBZ-containing proteins were targets of PARP1-mediated ADP-ribosylation dependent on a functional PBZ motif, and the analyzed PBZ-containing proteins co-localized with pADPr in vivo (Ahel et al., 2008). Importantly, a functional PBZ motif is essential for the role of CHFR in the antephase checkpoint and treatment with the PARP inhibitor KU- 0058948 overrides this checkpoint in CHFR-proficient cells (Ahel et al., 2008). Therefore, the PBZ motif might play a physiological role in DNA repair and cell cycle checkpoint processes dependent on its ability to recognize pADPr.

1.5 Insights into the catalytic mechanism of ADP-ribosylation reactions The structure of pADPr is well characterized (Figure 7): it is a homopolymer of ADP- ribose units linked by glycosidic ribose-ribose 2’-1’’ bonds (Chambon et al., 1966; Miwa et al., 1977; Reeder et al., 1967; Sugimura et al., 1967) with a heterogeneous chain length reaching up to 200-400 units in vitro and in vivo under conditions of stimulated DNA damage (Adamietz et al., 1978; Alvarez-Gonzalez and Jacobson, 1987; Juarez- Salinas et al., 1982). Long polymers synthesized by PARP1 and PARP2 can be branched in an irregular manner (Hayashi et al., 1983), and the chemical structure of the branching site was defined as O-D-ribofuranosyl-(1’’’-2’’)-O-D-ribofuranosyl-(1’’-2’)- adenosine-5’,5’’,5’’’-triphosphate (Miwa et al., 1981). Branching can occur with an average frequency of about one branching point per 20-50 linear ADP-ribose units (Alvarez-Gonzalez and Jacobson, 1987; Hayashi et al., 1983; Kawaichi et al., 1981; Miwa et al., 1979).

Figure 7. Structure of pADPr (from (Ruf et al., 1998b)). Displayed is the chemical structure of branched pADPr attached to a substrate protein via an ester linkage between the carboxylate group of an acidic amino acid side chain and the hydroxyl group of ribose (discussed above).

The conformation of long pADPr chains likely adapts a helicoidal structure resembling nucleic acids (Minaga and Kun, 1983a; Minaga and Kun, 1983b). Indeed, it was Introduction 26 observed that antibodies raised against pADPr show cross-reactivity with DNA and RNA, and vice versa (Kanai et al., 1978; Sibley et al., 1986; Sibley et al., 1988). Considering the complex chemical structure of pADPr, one might wonder about how a single enzyme might be able to catalyze all the different steps involved in the transfer of pADPr to target proteins. The following paragraph will try to shed light on the mechanistic details of the poly-ADP-ribosylation reaction catalyzed by PARP1. If necessary, short excursions to the mono-ADP-ribosylating toxins will help to understand certain catalytic details.

The process of poly-ADP-ribosylation by PARP1 can be subdivided into three parts: the initiation, the elongation and the branching reaction. The initiation is characterized by the attachment of a single ADP-ribose moiety from NAD+ to the -carboxy group of glutamic acid residues or less likely aspartic acid residues resulting in the formation of a labile ester bond that is unstable in dilute alkali and neutral hydroxylamine (Burzio et al., 1979; Kawaichi et al., 1980; Riquelme et al., 1979). The elongation step involves the transfer of further ADP-ribose moieties to an already attached ADP-ribose unit through glycosidic linkages (Chambon et al., 1966; Miwa et al., 1977; Ueda et al., 1979). The third reaction catalyzed by PARP1 is the branching reaction that occurs in an irregular manner (Kawaichi et al., 1981; Miwa et al., 1979; Reeder et al., 1967). In addition, at nanomolar NAD+ concentrations an abortive NAD+ glycohydrolase activity was suggested for PARP1 (Bauer et al., 1986). PARP1 undergoes a strong auto- modification reaction that targets several acidic residues residing mainly in the central auto-modification domain (Kawaichi et al., 1981). Although up to 28 auto-modification sites were reported (Desmarais et al., 1991; Kawaichi et al., 1981), analysis of the auto-mono-ADP-ribosylation by PARP1 suggests that a maximum of eight residues within the 22 kDa auto-modification domain are targeted (Mendoza-Alvarez and Alvarez-Gonzalez, 1999). The auto-modification likely exhibits an inhibitory effect on its catalytic properties, since an increase in Vmax and a decrease in Km was observed for the auto-modified enzyme compared to unmodified PARP1 (Kawaichi et al., 1981; Zahradka and Ebisuzaki, 1982). It has been reported that PARP1 itself is the major acceptor of pADPr in vitro (Kawaichi et al., 1981) as well as in vivo (Adamietz, 1987; Kreimeyer et al., 1984; Ogata et al., 1981). Accordingly, most of the enzymatic studies for PARP1 have analyzed the auto-modification reaction.

+ The Km values of purified PARP1 for NAD was determined to be ca. 20-80 M (Alvarez-Gonzalez, 1988; Kawaichi et al., 1981; Lagueux et al., 1995; Mendoza-

Alvarez and Alvarez-Gonzalez, 1993) with a Vmax of 0.5-2 pmol/min/mg enzyme and a Introduction 27

4 turnover rate kcat/Km 2.2 x 10 for the auto-poly-ADP-ribosylation reaction (Alvarez- Gonzalez, 1988; Kawaichi et al., 1981; Mendoza-Alvarez and Alvarez-Gonzalez, 1993). Histones were reported to act as allosteric activators of enzymatic PARP1 activity stimulating it up to 20-fold (Carter and Berger, 1982; Ito et al., 1979; Okayama et al., 1977; Petzold et al., 1981). In addition, a stimulation by some transcription factors was observed (Griesenbeck et al., 1999; Oei and Shi, 2001), with the mechanistic details remaining unsolved. Compared to the polymerization reaction, the initiation reaction + proceeds around 230 times slower with an apparent Km for NAD of around 12 M and 3 with a 50-fold lower kcat/Km 0.4 x 10 (Mendoza-Alvarez and Alvarez-Gonzalez, 1999). Thus, the initiation reaction is the rate-limiting step in the enzymatic reaction catalyzed by PARP1. It was suggested that the initial mono-ADP-ribosylation occurs in a distributive manner (Mendoza-Alvarez and Alvarez-Gonzalez, 1993), i.e. that potential target glutamates are mono-ADP-ribosylated in a random fashion without proceeding directly to the elongation reaction. In contrast, the polymerization reaction (elongation and branching) was shown to be highly processive (Naegeli et al., 1989), i.e. that once the enzyme has started the polymerization reaction at one mono-ADP-ribosylated residue, it will proceed until the possible longest and branched pADPr is synthesized onto this residue. The processive mode implies a constant pattern of reaction intermediates during the time course of the reaction. Indeed, it was observed that the relative size distribution of the synthesized pADPr remained constant during the auto- modification of PARP1, and in addition, the sequential polymerization led to a time- dependent increase in pADPr-carrying PARP1 molecules (Naegeli et al., 1989). Thus, PARP1 uses a processive mode for its polymerization reaction. The elongation reaction by PARP1 could theoretically occur via a protein-proximal mechanism or a protein- distal mechanism. Although results from early studies favored a protein-proximal mechanism (Ikejima et al., 1987), it was later shown that PARP1 uses a protein-distal mechanism for the elongation, adding ADP-ribose moieties to the AMP terminus of the growing pADPr chain (Alvarez-Gonzalez, 1988).

Several lines of evidence indicate that PARP1 acts as a homodimeric enzyme. First, using several biochemical approaches Bauer et al. (Bauer et al., 1990) were able to show that PARP1 associates in macromolecular complexes. The ability for self- association is encoded in the N-terminal part of PARP1 comprising the DBD and the auto-modification domain (Bauer et al., 1990). Furthermore, maximal catalytic activity correlated with the presence of dimeric forms of PARP1, while monomeric PARP1 was suggested to act predominantly as NAD+ glycohydrolase (Bauer et al., 1990). In support of these results, the central auto-modification domain was proposed to be Introduction 28 involved in the homo-/hetero-dimerization of PARP1 (Uchida et al., 1993). Second, it was shown that the PARP1 auto-modification reaction occurs intermolecularly, since initial rates of auto-poly-ADP-ribosylation and of initial auto-mono-ADP-ribosylation vs. the enzyme concentration increased with second order kinetics (Mendoza-Alvarez and Alvarez-Gonzalez, 1993; Mendoza-Alvarez and Alvarez-Gonzalez, 1999). Third, it was suggested that the binding of PARP1 to nicked dsDNA triggers its dimerization dependent on its DBD resulting in its subsequent enzymatic activation, promoting both the initiation as well as the elongation and branching reaction (Le Cam et al., 1994; Mendoza-Alvarez and Alvarez-Gonzalez, 1999; Panzeter and Althaus, 1994). This is supported by the observation that PARP1 protects SSBs in DNA footprinting assays in a symmetrical manner indicating that PARP1 binds nicked DNA as a dimer (Gradwohl et al., 1990; Menissier-de Murcia et al., 1989). Finally, proteolytic fragments of PARP1 can be modified in trans by the full-length protein pointing at an intermolecular auto- modification reaction (Kameshita et al., 1986). In summary, it is undisputed that the PARP1 auto-modification reaction needs a catalytic dimer, but it remains obscure if both molecules of the dimer function simultaneously as catalyst and acceptor or if one catalyzing molecule modifies one acceptor molecule. Regarding the high-molecular nature of pADPr and the potential sterical problems that might occur if two poly-ADP- ribosylated proteins have to be placed in a catalytically competent position to each other, it is tempting to speculate that the second mechanism is in place, although experimental evidence is still missing.

The basal enzymatic activities including initiation, elongation, branching and NAD+ glycohydrolase activity depend on the presence of the C-terminal 54 kDa catalytic domain of PARP1 comprising aa 526-1014 (Kameshita et al., 1986; Kameshita et al., 1984). Although initial reports suggested that the catalytic domain per se is not able to undergo auto-ADP-ribosylation and is enzymatically inactive if separated from the rest of the protein (Kameshita et al., 1986; Kameshita et al., 1984), Simonin et al. unambiguously showed that even a shorter 40 kDa C-terminal fragment of PARP1 retained in principle all the catalytic activities observed for the full-length protein except of the dramatic stimulation by DNA (Simonin et al., 1993a; Simonin et al., 1990). While + the Km value for NAD was comparable to the one determined for full-length PARP1 indicating equally efficient NAD+ binding, the specific activity was about 500-fold lower for the catalytic 40 kDa fragment similar to the basal activity of PARP1 in the absence of nicked DNA (Simonin et al., 1993a). Since the auto-modification domain that contains most if not all target glutamates for the auto-ADP-ribosylation by PARP1 is absent in the 40 kDa fragment, distinct glutamate residues must be targeted in this Introduction 29 auto-modification reaction. This can be interpreted as a rather unspecific targeting of surface-exposed glutamate residues by the catalytic domain of PARP1, an assumption in accordance with the broad range of substrates identified for PARP1 without any obvious consensus sequence. Thus, it is tempting to speculate that the distributive initiation reaction occurs rather unspecifically at glutamate residues of target proteins that are recruited, most likely by specific interactions with PARP1, in vicinity of the catalytic PARP1 domain. A similar mechanism was recently suggested for the histone acetyl transferases CBP/p300 which also target a broad range of substrates at lysine residues lacking any consensus sequence (Liu et al., 2008).

A first important step to determine the important residues involved in the catalysis by PARP1 was made by Marsischky et al. who aligned the catalytic domain of PARP1 to the catalytic domains of ADP-ribosylating toxins and mutated the most strikingly conserved residues (Marsischky et al., 1995). They suggested a similar active site structure between ADP-ribosylating toxins and PARP1 and observed a strong conservation of a histidine, a tyrosine and a glutamate residue in the catalytic domain of PARP1 from various species (Marsischky et al., 1995). Importantly, the corresponding amino acids in Diphtheria toxin were shown to participate in NAD+ binding (histidine and tyrosine) and directly in catalysis (glutamate) as extracted from the crystallographic structure (Domenighini et al., 1994). Indeed, replacing the conserved histidine with an alanine dramatically decreased the specific enzymatic activity rendering the enzyme in principle inactive (Marsischky et al., 1995). A comparable severe phenotype was observed for the mutation of the conserved Glu-988 + to an alanine. Although the Km for NAD was only little affected by this replacement, the

Vmax value were dramatically lower than in the WT enzyme resulting in an about 1,100- fold reduction in specific enzymatic activity (Marsischky et al., 1995). Careful enzymatic experiments revealed that the initiation reaction was reduced approximately 30-fold in the E-988A mutant, while the capacity to catalyze the elongation reaction was abrogated (Marsischky et al., 1995). A conservative replacement of Glu-988 with an aspartic acid residue resulted in retention of the elongation capacity, although the average chain length was about 2-fold shorter and the initiation reaction was impaired as seen for the E-988A mutant (Marsischky et al., 1995). Consistent with these observations, the authors proposed that the catalytic glutamate Glu-988 might act as a general base catalyst in the initiation reaction by activating the 2’-hydroxyl group of the terminal adenosine allowing its nucleophilic attack on the nicotinamide-ribose bond of an incoming NAD+ (Marsischky et al., 1995).

Introduction 30

In support of these results, the crystallographic structure of the catalytic PARP1 domain from chicken revealed significant structural similarities between PARP1 and ADP- ribosylating toxins (Ruf et al., 1996). The NAD+ binding motif of PARP1 differs significantly from the Rossman fold found in other NAD+-consuming enzymes (Figure 8). Its core is composed of five ß-strands arranged in two abutting ß-sheets and is complemented by a sixth ß-strand participating in this anti-parallel confirmation. An - helix is interspersed between ß-strands 2 and 3 and a long loop region connects ß- strands 4 and 5 (Otto et al., 2005; Ruf et al., 1996). By analyzing the PARP1 structure with a bound nicotinamide analogue and superimposing the results to the situation in bacterial toxins, a putative NAD+ binding site was determined to be between the two central ß-sheets with Gly-863, Tyr-907 and Glu-988 involved in binding the nicotinamide moiety and participating in catalysis (Ruf et al., 1996).

Figure 8. Structural comparison of the catalytic cores of PARP1 and Diphtheria toxin reveals a shared NAD+ binding motif (newly-arranged from (Otto et al., 2005)). For explanations, see top of next page. Introduction 31

Legend Figure 8: Two abutting sheets of anti-parallel strands form the upper and lower jaws of a Pacman-like NAD-binding crevice in all known structures of ADP-ribosyltransferases (upper panel). The structures are depicted from the "front view" with a full view of the ligands bound in the active site crevice. The ligands NAD and 3MB are colored cyan and are depicted as stick models. The central four -strands (from top to bottom: 5, 2, 1, 3, colored orange) are conserved in all mARTs and PARPs. The strands at the edges of the respective sheets ( 4 and 6, colored pink) show greater structural variation than the central strands. The H-Y-E motif residues are depicted in red and their side chains are shown as sticks. The glutamic acid residue (E-988 inPARP1) at the front edge of 5 is the critical catalytic residue in both Diphtheria toxin and PARP1. Diphtheria toxin (DT) and PARP1 share the following additional structural features: the orientation of 6, the alpha helix between 2 and 3 (colored yellow) and the conserved histidine (His-862 inPARP1) and tyrosine (Tyr-896 in PARP1) amino acid residues in 1 and 3. Note that the loop between 4 and 5 (colored magenta) differs significantly between both enzymes (discussed below). 3MB, 3-Aminobenzamide, an NAD-analogous PARP inhibitor

The central role of this catalytic core domain was corroborated by deletion experiments. As expected, removal of structurally conserved regions led to complete abrogation of the catalytic activity (Cherney et al., 1991; Simonin et al., 1990). Photoaffinity labeling identified two potential adenine binding residues, Trp-1014 at the extreme C-terminus and Lys-893 (Kim et al., 1997). While non-conservative mutations of Lys-893 abrogated enzymatic activity (Kim et al., 1997; Simonin et al., 1993b), Trp- 1014 was dispensable and seems not to play an important role in NAD+ binding (Kim et al., 1997; Simonin et al., 1993b). The refined structures of the inhibitor-bound and unligated catalytic PARP1 domain and a local structural alignment to the situation in bacterial toxins revealed further residues involved in NAD+ binding (Figure 9) (Ruf et al., 1998a).

Figure 9. Schematic drawing of hydrogen bonds (dotted lines) and hydrophobic contacts (parallel lines) to NAD+ (from (Ruf et al., 1998a)). Dashed-line boxes are used for residues from the N-terminal domain. In the homologous complex DT:NAD+ the phosphates are hydrogen bonded to water molecules, one of which is also present in unligated PARP-CF and depicted here. DT, Diphtheria toxin; PARP-CF, PARP1 catalytic fragment Introduction 32

It could be shown that the adenine moiety is buried in a deep pocket that leaves some space for water molecules and is fixed by several hydrogen bonds and hydrophobic interactions (Ruf et al., 1998a). Confirming previous results, the nicotinamide moiety is mainly hold in place by Gly-863 and Tyr-907 (Ruf et al., 1998a). Mutagenic studies confirmed the catalytic importance of residues located in the NAD+ binding motif comprising aa 855-1005 in PARP1 (Rolli et al., 1997). Mutation of many of these residues did neither effect the elongation nor the branching reaction, but decreased the specific enzymatic activity, which is in line with their potential role in binding the co- substrate NAD+. Interestingly, some of the catalytically important residues are not located in the catalytic core domain, but either in the N-terminal -helical domain (aa 662-784) or in the external face of the catalytic domain (Rolli et al., 1997). Due to the occlusion of the adenine binding pocket by helix F and the close vicinity of some residues to the active site, the N-terminal helical domain was suggested to relay the DNA binding signal to the catalytic domain resulting in a rearrangement more favourable for catalysis (Ruf et al., 1996). Indeed, the gain-of-function mutant L713F + residing in helix D exhibits a 9-fold higher activity, while the Km for NAD is unchanged (Miranda et al., 1995). Leu-713 is positioned near aa 881-888 which possess low mobility and are equivalent to the “active site loop” in bacterial toxins (Ruf et al., 1998a). In contrast to the situation in PARP1, this loop becomes labile upon NAD+ binding in the toxins (Bell and Eisenberg, 1996; Li et al., 1996). It was suggested that the helix D participates in mobilization of this region upon DNA-binding by PARP1, thereby increasing the access to the substrate (Rolli et al., 1997). Besides these findings, the random mutagenesis of residues in the catalytic domain of PARP1 revealed that especially Tyr-986 close to the catalytic glutamate Glu-988 participates in the elongation reaction (Rolli et al., 1997). While the conservative Y986S mutant was only slightly affected in the elongation reaction, the non-conservative substitution Y986H showed in addition an about 15-fold increase in branching frequency (Rolli et al., 1997). The production of short and highly branched pADPr by PARP1 Y986H indicates that branching is not per se restricted to longer ADP-ribose chains. Instead, it was suggested that the intrinsic helical nature of longer pADPr presents a more favorable conformation for branching than the shorter linear forms, and that the mutant Y986H would introduce a distortion of the polymer terminus positioning the terminal ribose in a convenient manner (Rolli et al., 1997).

As outlined above the bacterial mono-ADP-ribosyltransferases and PARP enzymes share a similar NAD+ binding fold and a catalytic glutamate residue at a conserved position in the vicinity of the nicotinamide ribose (Domenighini et al., 1994; Domenighini Introduction 33 and Rappuoli, 1996; Okazaki and Moss, 1994; Ruf et al., 1998a). An essential role for the conserved glutamate has repeatedly been demonstrated for both the toxins (Aktories et al., 1995; Bohmer et al., 1996; Douglas and Collier, 1990; Lukac and Collier, 1988; Pizza et al., 1988) and PARP1 (Marsischky et al., 1995). The catalytic glutamate was proposed to act as a general base catalyst to remove a hydrogen from the incoming nucleophilic ribose (Marsischky et al., 1995), but studies from bacterial toxins showed that it acts by stabilizing the transition state of the reaction: the charged nicotinamide of NAD+ is bound in a hydrophobic pocket and isolated from solvent, which promotes the transition state by ground state destabilization (Berti et al., 1997). The C1’ carbon faces out of the active site, poised for attack by the incoming nucleophile, which can be a diphthamide moiety, arginine, cysteine, or glutamic acid, depending on the identity of the ADP-ribosyltransferase. Reaction of the nucleophile with C1’ leads to an inverted configuration in the ADP-ribosylated product (Oppenheimer, 1978; Oppenheimer and Bodley, 1981; Scheuring and Schramm, 1995). For the toxins, kinetic isotope effects of the enzyme-catalyzed hydrolysis of NAD+ as well as ADP-ribosylation reactions indicate that the transition state bond orders are small for both the nicotinamide leaving group and the incoming nucleophile, and that the transition state has a high degree of oxocarbenium ion character (Berti et al., 1997; Parikh and Schramm, 2004; Rising and Schramm, 1997; Scheuring et al., 1998; Scheuring and Schramm, 1997). In other words, when the nucleophile has begun to form a bond with the anomeric carbon (C1’), the bond to the leaving group has almost been broken. Thus, for diphtheria toxin, the NAD+ hydrolytic reaction has been termed “dissociative Sn2” (Berti et al., 1997). The catalytic glutamate does not appear to be important for binding of NAD+, since its mutation in PARP1 (Marsischky et al., 1995) as well as bacterial toxins (Bohmer et al., 1996; Douglas and Collier, 1990) does not affect the dissociation constant for NAD+. Instead, the catalytic glutamate functions to stabilize the oxocarbenium ion transition state that develops during the reaction. The intimate contact between the nicotinamide ribose and the conserved glutamic acid has been demonstrated by photoaffinity labeling studies (Aktories et al., 1995; Barbieri et al., 1989; Carroll and Collier, 1984; Carroll and Collier, 1987), which have shown that the gamma carbon of the glutamate side chain becomes covalently linked to C6 of the nicotinamide ring (Carroll et al., 1985). On this basis, the carboxyl group of the catalytic glutamate must be very close to the C1’-N1 bond, and therefore well-positioned to stabilize the oxocarbenium ion transition state through a charge interaction. The oxocarbenium of the transition state forces the four ribose atoms C2’, C1’, O4’ and C4’ of the nicotinamide ribose into a planar conformation. Since the 3’- endo conformation of the nicotinamide ribose is close to this transitional conformation Introduction 34 and the 2’-endo conformation shields the C1’ atom, the bacterial mono-ADP- ribosyltransferases bind NAD+ in this favorable conformation (Bell and Eisenberg, 1996; Li et al., 1996). The same was assumed to hold true for PARP1 (Ruf et al., 1998a). A recent study analyzed the role of Glu-988 in the PARP1-mediated hydrolysis of NAD+ by means of a combined quantum mechanical/molecular mechanical (QM/MM) study. As demonstrated for the bacterial toxins, it could be shown that the reaction indeed proceeds through a SN2 dissociative mechanism and involves an oxocarbenium ion transition state (Bellocchi et al., 2006). The catalytic Glu-988 stabilizes the Michaelis complex of the reaction and the transition state via electrostatic interaction, thereby lowering the activation energy by 13.4 kcal mol-1 (Bellocchi et al., 2006). Thus, the essential importance of the highly conserved catalytic glutamate for the enzyme-catalyzed ADP-ribosyltransferase reaction is undisputed.

Based on the previously obtained results and on a crystal structure of PARP-CF with the non-hydrolysable carba-NAD+, Ruf et al. suggested the following reaction mechanism for the elongation and branching reaction by PARP1 (Figure 10): the proposed acceptor site binding the prolongable pADPr forms a unique groove which is absent in other ADP-ribosyltransferases (Ruf et al., 1998b). Especially the pyrophosphate of the terminal ADP-ribose is bound tightly through several hydrogen bonds, and the adenine moiety is hold in place by hydrophobic interactions with Met- 890. Importantly, the mutants K903Q and K903E lost their ability to form pADPr without affecting the binding of NAD+ confirming the important role of the acceptor site in pADPr formation and underlining that the non-affected initiation reaction requires structural conditions distinct from the ones for elongation (Ruf et al., 1998b). The NAD+ donor site was described previously from studies on inhibitor-bound PARP1-CF (Ruf et al., 1998a) and was confirmed by mutations targeting Gly-863, Tyr-896 and Tyr-907. As expected, these mutations diminished the binding of NAD+ without affecting the enzyme’s capacity to synthesize pADPr (Ruf et al., 1998b). In the elongation reaction the catalytic glutamate acts by increasing the nucleophilicity of the acceptor ribose and by stabilizing the oxocarbenium ion transition state in the donor nicotinamide ribose. Since in the initation reaction the nucleophile is a carboxylate group of an amino acid side-chain that under physiological pH does not require the activation by Glu-988 as base catalyst, the role of the catalytic glutamate in the initiation reaction is restricted to the stabilization of the transition state (Ruf et al., 1998b). For the branching reaction, a rotation of the bound pADPr by 180° was proposed, which might be possible due to the fact that the active site in PARP1 is open on both sides. In the reverse orientation, the nicotinamide ribose is bound to the acceptor site instead of the adenine ribose allowing Introduction 35 the catalysis of branched pADPr (Ruf et al., 1998b). In summary, data from these studies explain the role of the conserved His-Tyr-Glu triad in the discussed ADP- ribosyltransferases, with histidine and tyrosine participating in the binding of NAD+ and with the catalytic glutamate acting to stabilize the oxocarbenium transition state and promoting the elongation reaction by facilitating the nucleophilic attack of the ribose hydroxyl groups.

As outlined above, the enzymatic activity of mARTs and PARPs was described to be dependent on a well-conserved catalytic glutamate residue in the active center (Bellocchi et al., 2006; Holbourn et al., 2006; Marsischky et al., 1995; Ruf et al., 1998b). However, in several of the novel postulated PARP enzymes the existence of this important glutamate is disputed by sequence comparisons, suggesting that either these enzymes are catalytically inactive or that another mechanism is in place (Ame et al., 2004; Otto et al., 2005). Thus, these findings indicate that the PARP family may not be homogenous regarding their catalytic activity. Instead it is possible that distinct subfamilies exist that may differ in their enzymatic activity as well as other features.

Figure 10. Proposed reaction mechanism for the elongation and branching reaction by PARP1 (from (Ruf et al., 1998b)). Left panel: Schematic drawing showing hydrogen bonds as dotted lines and nonpolar interactions as parallel bars. The nucleophilic attack is marked by an arrow. Right panel: A polymer subunit consists of A (adenine ribose), P (phosphate), N (nicotinamide ribose) and ad (adenine). Apart from the adenine, the subunit shows internal symmetry. The binding sites for the donor NAD+ and for the accepting subunit of pADPr are marked by dotted rectangles. At the acceptor site, the adenine is only weakly bound when compared with the phosphates, and there is space for polymer extensions over both ends of the site. Consequently, the polymer can bind in two orientations related by a 180° rotation (left and right sketch). Because of the given donor site position, the two binding orientations at the acceptor site give rise to elongation and branching as sketched. Introduction 36

1.6 Protein sorting: directing proteins to the desired destinations Regarding the fact that several thousand proteins coexist in a single cell, each occurring in copies from a few hundreds to several millions, it is obvious that specific proteins have to be targeted efficiently to their destinations within the cell. This targeting is guided by sorting signals in the transported protein. One can distinguish three general classes of sorting signals: signal sequences representing a continuous stretch of amino acids that are often removed from the mature protein by a signal peptidase, signal patches representing the three-dimensional arrangement of amino acids from different regions of the primary protein sequence, and post-translational modifications like glycosylation or fatty acylation. Proteins are targeted via these sorting signals to different destinations: to cytoplasmic organelles like mitochondria, lysosomes or peroxisomes, to the nucleus, and in many cases to the ER and the Golgi where especially secreted and membrane proteins pass the secretory pathway. With respect to this work, the transport across the nuclear envelope will be described in more detail.

1.7 Transport across the nuclear envelope The double lipid bilayer of the nuclear envelope separates the nucleus and the cytoplasm and represents a significant barrier for the export of RNAs and proteins into the cytoplasm as well as for the nuclear import of proteins. The transport across the nuclear envelope occurs via nuclear pore complexes (NPCs) that form channels spanning the nuclear envelope. NPCs are complex macromolecular structures of 60- 130 MDa composed of several copies of about 30 different proteins called nucleoporins (Allen et al., 2000). The NPC consists of several ring systems and additional components, and the majority of NPCs shows a typical eight-fold radial symmetry (Figure 11) (Allen et al., 2000).

Introduction 37

Figure 11. Structure of the nuclear pore complex (from (Allen et al., 2000)). Shown is a structural representation of the NPC with direct visualization of individual components. The presence of a central transporter is disputed due to experimental evidence arguing against it (Fahrenkrog and Aebi, 2003).

The functional inner diameter of the nuclear pore is around 30 nm allowing ions, metabolites, and proteins smaller than 40-60 kDa to diffuse across the nuclear envelope (Allen et al., 2000; Terry et al., 2007). But the majority of the proteins have to be transported in an active fashion via interaction with specialized import and export proteins called karyopherins, which can be subdivided into importins and exportins regarding their function (Pemberton and Paschal, 2005). These interactions are driven via signal sequences termed nuclear localization signal (NLS) or nuclear export sequence (NES) (Macara, 2001). The classical NLS, which is the predominant signal to target proteins to the nucleus, is a monopartite or bipartite stretch of basic amino acids and is recognized by importin- (Lange et al., 2007). Many proteins entering the nucleus lack a classical NLS and can be imported e.g. through a direct interaction with importin-ß, through a piggyback mechanism along with an interacting NLS-containing protein or through a basic surface patch functioning in an NLS-like fashion (Macara, 2001; Stewart, 2007). The classical NES has a Leu-rich consensus sequence and is less clearly defined as the NLS because other hydrophobic sequences can replace the conserved leucines and the length of the intervening stretches are partially variable Introduction 38

(Macara, 2001). The NES mediates the interaction of the NES-harboring protein with the export protein Crm1 (Pemberton and Paschal, 2005).

Common to all nucleocytoplasmic shuttling processes, regardless of whether import or export is in place or if the transport is mediated by classical NLS or NES, is the dependency on the small GTPase Ran. Most of the Ran molecules are present in their GTP-bound state and are localized in the nucleus, but a small subpopulation of Ran- GDP is cytoplasmic. The gradient of Ran-GTP between the nucleus and the cytoplasm is the driving force of the nuclear import/export processes, and in addition, Ran-GTP is responsible for the disassembly of import cargo complexes and the assembly of export cargo complexes (Figure 12) (Macara, 2001; Pemberton and Paschal, 2005).

The Ran gradient is maintained through important regulators of Ran, RanGAPs and RanGEFs. RanGAPs activate the intrinsic GTPase function of Ran resulting in the hydrolysis of GTP to GDP, while RanGEFs act as nucleotide exchange factors converting Ran-GDP to Ran-GTP (Quimby and Dasso, 2003). The nuclear RanGEF RCC1 is associated with chromatin and efficiently maintains the high nuclear levels of Ran-GTP, while RanGAP is localized to the cytoplasmic face of the NPC and activates the GTP hydrolysis leading to cytoplasmic Ran-GDP (Quimby and Dasso, 2003). Thus, Ran and its respective GEF and GAP play an essential role in transport processes across the nuclear envelope.

Introduction 39

Figure 12. Overview about some major nuclear transport pathways (from (Pemberton and Paschal, 2005)). A. Nuclear import of Ran-GDP mediated by NTF2. B. Nuclear import of nuclear localization sequence (NLS) cargo mediated by the karyopherin-: importin-ß1 heterodimer (abbreviated Imp- and Imp-ß). Nuclear import of NLS cargo mediated by direct binding to importin-ß1 and other karyopherin-ß family members is not shown. C. Nuclear export pathways that mediate recycling of importin-ß1 and karyopherin-; the latter requires CAS as an export receptor. D. Nuclear export of nuclear export sequence (NES) cargo mediated by Crm1. RanGAP is anchored to the cytoplasmic side of the NPC and RanGEF is shown bound to chromatin. For simplicity, the model depicts only the minimal components necessary to form the pretranslocation transport complexes, and post-translocation intermediates and accessory factors are not shown. Introduction 40

1.8 Aim of this work

ADP-ribosylation represents an evolutionary ancient post-translational modification and serves as a regulator of protein function. The enzymes involved can be divided into mono-ADP-ribosylating enzymes including bacterial toxins and into the poly-ADP- ribosylating enzymes of the PARP family. It was described previously that PARP10 possesses ADP-ribosyltransferase activity and undergoes auto-modification. Although an antibody directed towards poly-ADP-ribose displayed weak reactivity towards ADP- ribosylated PARP10, the ability of PARP10 to form polymers of ADP-ribose has not been shown conclusively. Recent sequence alignments suggested that the PARP superfamily that is constituted of seventeen members is heterogenous regarding essential residues involved in catalysis. Since the presence of a conserved glutamate residue in the active center of PARP10 was disputed, we considered analyzing the catalytic activity of PARP10 in-depth. Therefore the first part of this work examines the catalytic properties of PARP10 and extends the findings to related enzymes of the PARP family.

PARP10 is a mainly cytoplasmic protein that accumulates in the nucleus upon treatment with Leptomycin B, a potent inhibitor of Crm1-dependent nuclear export. While a functional NES was identified in PARP10, no classical NLS mediating nuclear import could be determined. Thus it remained elusive how PARP10 is able to enter the nucleus. Since the PARP10-interacting protein c-MYC is an exclusively nuclear protein, understanding the nuclear import mechanism would help to gain insights into the functional interaction between PARP10 and c-MYC. Therefore the second part of this work analyzes the subcellular localization of PARP10 and its nucleocytoplasmic shuttling in order to define the regions responsible for nuclear uptake. Results and discussion 41

2. Results and discussion

2.1 The PARP10-mediated ADP-ribosylation reaction

2.1.1 PARP10 functions as mono-ADP-ribosyltransferase Our previous studies had shown that the C-terminal half of PARP10 possesses catalytic activity (Yu et al., 2005b). To further evaluate the enzymatic properties of PARP10, we purified full-length PARP10 and a catalytically inactive mutant (PARP10- G888W) by Tandem Affinity Purification (TAP) from HEK293 cells (Fig. 13A and data not shown). Mass spectrometry analysis and reactivity with PARP10-specific antibodies confirmed that these proteins are PARP10 (data not shown). ADP-ribosylation assays demonstrated robust C-TAP-PARP10 auto-modification with incorporation being linear over at least 20 minutes (Fig. 13B). However, C-TAP-PARP10 did not display a mobility shift upon auto-ADP-ribosylation, which is indicative of the pADPr formation catalyzed by PARP1, PARP2 and the Tankyrases (PARP5a and b) (Fig. 14) (Ame et al., 1999; Naegeli et al., 1989; Rippmann et al., 2002; Smith et al., 1998).

Figure 13. A. C-TAP-PARP10 and C-TAP were purified from stably transfected HEK293 cells and analyzed by Coomassie blue staining. B. Purified C-TAP-PARP10 was incubated with 32P-NAD+ and 50 M NAD+ at 30°C for the times indicated. The reactions were stopped by adding SDS-sample buffer and the proteins were analyzed by SDS-PAGE and autoradiography. The displayed experiment is representative for two independent experiments.

Therefore we compared the activities of PARP10 and PARP1. Purified C-TAP-PARP10 and N-TAP-PARP10 underwent auto-modification, while C-TAP-PARP10-G888W was Results and discussion 42 inactive (Fig. 14A). Importantly, neither C-TAP-PARP10 nor N-TAP-PARP10 revealed any change in mobility, unlike PARP1 that showed a substantial increase in apparent molecular weight due to pADPr formation (Fig. 14A). Furthermore and in contrast to PARP1, auto-modified PARP10 was not or only poorly stained by mAb 10H that was reported to recognize pADPr chains of at least twenty ADP-ribose moieties (Kawamitsu et al., 1984) (Figs. 14B and a 20-fold longer exposure in 14C). A weak signal was measurable by pAb 96-10-04 that is described to recognize oligomers of six or more ADPr moieties (Alexis Biochemicals) (Fig. 14B). In both situations no shift in apparent mobility of PARP10 was detected.

Figure 14. For legend, see top of next page. Results and discussion 43

A. Indicated proteins were incubated in the presence of a constant amount of 32P-NAD+ and increasing concentrations of unlabeled NAD+ as indicated. Proteins were separated by SDS- PAGE, visualized by Coomassie blue staining and incorporated label detected by autoradiography. B. The reactions were performed as in panel A but without 32P-NAD+. The reaction products were analyzed by Western blotting using polyclonal antibodies (96-10-04) or a mAb (10H) specific for pADPr. PARP1 and PARP10 were detected using specific polyclonal antibodies. C. The experimental set-up was as in Fig. 2B. The reaction products were analyzed by Western blotting using mAb (10H) specific for pADPr. The staining with mAb 10H was 20x longer as that in Fig. 14B. D. HEK293 cells were transfected with plasmids expressing the indicated HA-tagged proteins. These were immunoprecipitated from whole cell lysates and their auto-ADP-ribosylation measured using 32P-NAD+ and the indicated concentrations of unlabeled NAD+. E. Endogenous PARP10 was immunoprecipitated from U937 promyelocytes using the polyclonal serum 891-6 or the corresponding prebleed 891-0 (Yu et al., 2005b) and analyzed for auto-ADP-ribosylation as in panel B. All experiments were at least performed twice with similar findings.

To exclude interference of the residual TAP-tag with catalytic activity, we expressed HA-tagged versions of PARP10 and PARP1 transiently in HeLa cells. TAP- and HA- tags display no sequence homology. Analysis of the HA-tagged proteins supported the results obtained for TAP-tagged PARP10 (Fig. 14D). Finally endogenous PARP10 from U937 promyelocytes was immunoprecipitated using a polyclonal PARP10 antiserum (Yu et al., 2005b). Similarly, this protein auto-modified without any change in mobility (Fig. 14E) and was not reactive to mAb 10H (data not shown). Thus full-length PARP10 is unable to generate long polymers that would alter the mobility of the protein on SDS- gels.

The efficient catalysis of long, branched pADPr by PARP1 is stimulated by its interaction with DNA (D'Amours et al., 1999). Since PARP10 possesses potential RNA and/or DNA interaction motifs (Yu et al., 2005b), we tested whether nucleic acids could modify its catalytic activity. The addition of sheared salmon sperm DNA, linear or supercoiled plasmid DNA, or total cellular RNA neither stimulated the activity of full length PARP10 nor resulted in polymer formation (Fig. 15A and data not shown). Instead DNA led to a dose-dependent reduction of auto-modification. This was also true for GST-PARP10(818-1025), which contains the catalytic domain but not the N- terminal potential RNA/DNA interaction motifs, indicating a direct effect of DNA onto the catalytic domain (Fig. 15B). Furthermore we addressed whether PARP10 is capable of catalyzing pADPr formation on heterologous substrates. As shown for a bacterially expressed catalytic fragment of PARP10 (Yu et al., 2005b), all four core histones were substrates of C-TAP-PARP10 and GST-PARP10(818-1025) but did not show any obvious shift in apparent molecular weight (Fig. 15C). Results and discussion 44

Figure 15. A. C-TAP-PARP10 and C-TAP-PARP10-G888W were auto-modified. The reactions contained the indicated amounts of sonicated salmon sperm DNA and total cellular RNA from HeLa cells. B. The experimental set-up was as in panel A. Here bacterially expressed GST-PARP10(818- 1025) was used that contains the catalytic domain. C. C-TAP-PARP10, C-TAP-PARP10-G888W and GST-PARP10(818-1025) were used to ADP-ribosylate core histones or BSA as indicated.

To further compare the PARP1- and PARP10-catalyzed reactions, auto-modification products were analyzed directly, first by assaying for AMP and phosphoribosyl-AMP (PRAMP), and second by determining polymer length. PRAMP represents the phosphodiesterase reaction product of pADPr, while AMP is derived from mono-ADP- ribosylated proteins and the terminal ADP-ribose of polymers. Auto-modified PARP1 yielded a strong PRAMP signal, but PRAMP was not detectable in the PARP10- catalyzed reaction (Fig. 16A), consistent with a lack of pADPr formation on auto- modified PARP10. In contrast, AMP was released from auto-modified PARP10, confirming that it is mono-ADP-ribosylated. AMP was a minor product of auto-modified PARP1, consistent with long polymers. Indeed, PARP1 catalyzed the formation of Results and discussion 45 longer polymers with increasing NAD+ concentrations, while mainly monomeric ADP- ribose and minute amounts of di- and trimers were associated with PARP10 auto- modification (Fig. 16B). This difference in mono- vs. poly-ADP-ribose formation was reflected in the considerably lower Vmax of PARP10 as compared to PARP1 (2 vs 500 pmol/min/g, respectively) (Alvarez-Gonzalez, 1988). However, the KM values for NAD+, roughly 50 M for PARP10 (Fig. 16C), were comparable to the values determined for PARP1 (Mendoza-Alvarez and Alvarez-Gonzalez, 1993).

Figure 16. A. PARP1 and C-TAP-PARP10 were auto-modified under standard reaction conditions using 5Ci of 32P-NAD+. ADP-ribosylation reaction products were subjected to phosphodiesterase treatment. Digestion products were analyzed by 2D-TLC and detected by autoradiography. B. PARP1 and PARP10 were auto-modified as described in panel A. Reaction products were removed from the proteins by base treatment and analyzed on a sequencing gel. Arrowheads indicate the length of ADP-ribose chains as determined by comigration of bromophenol blue (8- mer) and xylene cyanol (20-mer) (Alvarez-Gonzalez and Jacobson, 1987; Tanaka et al., 1978). Autoradiograms are displayed. C. PARP10 auto-modification reactions were performed with a fixed amount of 32P-NAD+ and increasing concentrations of NAD+. The incorporated label was determined by TCA precipitation and Cerenkov counting and the data analyzed by Lineweaver-Burk. The displayed kinetic resulted from the mean values of three independent experiments.

In summary, the lack of detectable mobility shifts of auto-modified PARP10 and of ADP-ribosylated core histones, the poor reactivity with two antibodies commonly used to detect pADPr, the lack of PRAMP upon phosphodiesterase treatment, and the lack Results and discussion 46 of detectable polymer formation, suggest strongly that PARP10 possesses mono-ADP- ribosyltransferase but not polymerase activity.

2.1.2 Molecular base for the transferase activity of PARP10 A model for the PARP10 catalytic domain was generated using the recently determined crystal structure of PARP12 (PDB-ID 2PA9; Structural Genomics Consortium, unpublished), with which PARP10 shares 33% sequence identity. A structure-based alignment of PARP1, PARP3, PARP12, and the PARP10 model is shown in Figure 17.

Figure 17 (in collaboration with B.H. Shilton). A structure-based sequence alignment of the catalytic domains of PARPs 1, 3, 10, and 12 is shown. Yellow shadings indicate structurally conserved regions. Asterisks indicate the residues of the conserved “HYE” triad (see text for details).

PARP1 has pADPr polymerase activity, while PARP10 catalyzes only mono-ADP- ribosylation. To identify the origins of this difference we compared the PARP10 model with that of PARP1. Core secondary structure elements are retained (Fig. 18A), as are the histidine (H887) and tyrosine (Y919) residues involved in NAD+ binding, and which constitute the first two amino acids of the highly conserved “HYE” triad found throughout the ADPr transferase superfamily (Figure 18B). However, it is clear that in PARP10 an isoleucine (I987) has replaced the catalytic glutamate found in PARP1 (E988) (Fig. 18B). Therefore, even though a catalytic glutamate is present in all other related ADP-ribosyltransferases (Domenighini et al., 1994; Domenighini and Rappuoli, 1996; Okazaki and Moss, 1994), the structure of PARP12 and the PARP10 model confirm the surprising observation that the catalytic glutamate is not present in these enzymes. Perhaps more surprising is that a close inspection of the PARP10 and PARP12 structures indicates there is no other residue in the active site that could substitute for the catalytic glutamate.

Results and discussion 47

Figure 18 (in collaboration with B.H. Shilton). A. Superposition of PARP1 (magenta) and the PARP10 model (blue); only the backbone CA (C alpha) atoms are shown. The expected position of bound NAD+ is indicated based on the structure of diphtheria toxin in complex with NAD+ (Bell et al., 1996). Secondary structure elements are labeled and correspond to the structurally conserved regions indicated in Figure 17. In addition, the side chain of I987 in PARP10 is shown; this residue is at the beginning of -5, in the position occupied by the catalytic glutamate in PARP1. A stereoview is displayed. B. Detailed view of the nicotinamide-ribose binding site in PARP1 (magenta) and PARP10 (blue). The side chains of the “HYE” motif in PARP1 (residues H862, Y896, and E988) are shown, along with the corresponding residues in PARP10. A stereoview is displayed.

In addition to the absence of the catalytic glutamate in PARP10, there is a striking difference in the sequence that connects ß-4 and ß-5. In PARP1, this connecting loop is 37 residues long, whereas it is only 6 residues long in PARP10 (Fig. 17). In PARP1 the catalytic glutamate (E988) is positioned at the N-terminus of ß-5, and the length and structure of the ß-4/ß-5 loop makes the region around E988 relatively crowded and constricted. In contrast, the short loop in PARP10 adopts a conformation that Results and discussion 48 effectively removes it from the active site region, making it much more open and accessible when compared with PARP1 (Fig. 18A).

The lack of a glutamate in the catalytic site of PARP10, as well as the structure of the ß-4/ß-5 loop, which has been previously implicated in substrate recognition (Han and Tainer, 2002; Ruf et al., 1998b; Sun et al., 2004), appear to be the two features of PARP10 that correlate with its lack of pADPr polymerase activity. A comprehensive sequence alignment (Otto et al., 2005), indicates that PARPs 6 through 16 also lack a catalytic glutamate, and, in common with PARP10, most of these enzymes have a 6- residue ß-4/ß-5 loop with a proline two residues N-terminal to the position where the catalytic glutamate is normally found. We have defined three PARP subfamilies based on the HYE triad at the catalytic core (Fig. 17, asterisks) and the length of the ß-4/ß-5 loop (Table 1). The first is characterized by an HYE triad and a long ß-4/ß-5 loop. This subfamily consists of PARPs 1-5, with PARPs 1, 2, 5a and 5b having been demonstrated to synthesize polymers. In the second group (PARPs 6-8, 10-12, and 14- 16) the HYE motif is replaced by HYI/L/Y and the loop is considerably shorter. The first member of this group that has been analyzed in detail is PARP10, reported here. Accordingly we suggest that the members of this group function only as mono-ADP- ribosyltransferases rather than polymerases. PARPs 9 and 13 of the third group possess QYT or YYV triads, respectively. These two PARP family members lack the catalytically important glutamate and the histidine, which binds the ß-NAD+ cofactor (Ruf et al., 1996; Ruf et al., 1998b). Since PARP9 has been reported to be catalytically inactive (Aguiar et al., 2005), we suggest that PARP13 will most likely also be inactive.

To evaluate these hypotheses we tested the activities of different catalytic domains. The catalytic domain of PARP1, PARP1cat, although poorly active in comparison to the full-length protein, was able to catalyze the formation of long pADPr polymers, as indicated by a massive shift of the ADP-ribosylated protein to higher molecular weight and strong staining with mAb 10H (Fig. 19A and data not shown). The catalytic domain of PARP13, with YYV in the catalytic center and thus lacking both the glutamate and the histidine, did not reveal any auto-ADP-ribosylating activity (Fig. 19A). PARP14 was capable of ADP-ribosylation, but did not reveal any mobility shift, did not react with mAb 10H, and upon phosphodiesterase treatment only AMP but no PRAMP was released (Figs. 19A, B and data not shown). Thus PARP14 functions as a mono-ADP- ribosyltransferase and not a polymerase. It is important to note that the lack of observed auto-ADP-ribosylation by PARP13 cannot only be explained by a lack of catalytic activity but also by the absence of potential auto-modification sites in the Results and discussion 49 analyzed GST fusion protein. Therefore an accurate analysis of the enzymatic properties of full-length PARP13 is necessary to draw a clear conclusion about its catalytic capacity.

Figure 19. A. The catalytic domains of human PARP13 and murine PARP14 were amplified and expressed as GST-fusion proteins. Auto-modification activities of these two proteins and of the baculo- expressed catalytic domain of PARP1 (aa656-1014) were analyzed by incorporation of 32P- ADP-ribose with increasing NAD+ concentrations. B. GST-mPARP14 was auto-modified under standard reaction conditions using 5Ci of 32P- NAD+ and 50 M NAD+ at 30°C for 30 min. ADP-ribosylation reaction products were detached from TCA-precipitated proteins by alkaline treatment, extracted with phenol/chloroform and subjected to phosphodiesterase treatment. Digestion products were analyzed by 2D-TLC and detected by autoradiography. The displayed experiments were performed twice with similar results.

The above findings support the structure- and sequence-based division of the PARP family into three functionally distinct subgroups, one with pADPr polymerase activity, one with mono-ADP-ribosyltransferase activity and one that is catalytically inactive (for summary see Table 1). Further studies analyzing the enzymatic properties of members of these subgroups will show if the proposed classifications can endure or if they will have to be re-adapted.

Results and discussion 50

Table 1. Comparison of catalytic core motifs and loop length (between ß-sheets 4 and 5) and described/suggested enzymatic activities of PARP family members.

PARP Protein Gene Catalytic Loop Postulated Demonstrated Demonstrated Demonstrated name symbol core length ADP- auto-ADP- polymerase mono-ADP- motif1 (between ribosylation ribosylation activity ribose ß-sheets 4 activity transferase and 5)2 (mono (M) activity vs poly (P)) 1 PARP1 PARP1 HYE +++ (37) P + + - (PARP) 2 PARP2 PARP2 HYE +++ (40) P + + - 3 PARP3 PARP3 HYE +++ (42) P + ? - 4 VPARP PARP4 HYE ++ (14) P + + (short - polymers) 5a TANK1 TNKS HYE ++ (10) P + + (no - branching) 5b TANK2 TNKS2 HYE ++ (10) P + + (no - branching) 6 PARP6 PARP6 HYI + (2) M ? ? ? 7 TiPARP TIPARP HYI + (6) M + ? ? 8 PARP8 PARP8 HYI + (2) M ? ? ? 9 BAL1 PARP9 QYT + (6) inactive - - - 10 PARP10 PARP10 HYI + (6) M + - (this study) + (this study) 11 PARP11 PARP11 HYI + (6) M ? ? ? 12 PARP12 ZC3HDC1 HYI + (6) M ? ? ? 13 PARP13 ZC3HAV1 YYV + (6) inactive - (this study) - - 14 BAL2 PARP14 HYL + (6) M + - (this study) + (this study) 15 BAL3 PARP15 HYL + (6) M + ? ? 16 PARP16 PARP16 HYY ++ (13) M ? ? ?

1Three amino acids relevant for catalytic activities of PARP enzymes. Histidine and tyrosine are within the catalytic center and important for function. The third amino acid, glutamate, has been demonstrated to be important for polymerase activity of PARP1 (Marsischky et al., 1995; Rolli et al., 1997). The three postulated subfamilies, i.e. the pADPr polymerases (yellow), the mono- ADP-ribosyltransferases (green), and the catalytically inactive PARP members (grey), are indicated. 2 +++, ++, and + indicate long, intermediate and short loops, respectively.

2.1.3 Characterization of the catalytic activity of PARP10: ADP-ribosylation of acidic residues To understand the transferase function of PARP10 in more detail, we analyzed its catalytic properties. The auto-modification of PARP10 proceeded with second order kinetics, indicative of an intermolecular reaction (Fig. 20A). In addition the inactive C- TAP-PARP10-G888W was a substrate of GST-PARP10(818-1025), which contains the catalytic domain, supporting the notion that the reactions occur intermolecularly (Fig. 20B). This is in accordance with the results obtained for PARP1 (Kameshita et al., 1986; Mendoza-Alvarez and Alvarez-Gonzalez, 1993). In order to define the region in PARP10 that is ADP-ribosylated, we prepared overlapping GST fusion proteins covering PARP10. When incubated in the presence of C-TAP-PARP10, the only modified fragment was GST-PARP10(818-1025) (Fig. 20B). Thus, this fragment is not only catalytically active but it also contains the auto-modification site(s). Results and discussion 51

Figure 20. A. C-TAP-PARP10 was titrated as indicated and the reaction products analyzed by SDS-PAGE (left panel). The labeled bands were cut out from the dried gel and counted. The mean values and standard deviation of three experiments are summarized in the right panel. B. The indicated PARP10 fragments were expressed as GST-fusion proteins and subjected to an enzymatic PARP assay with C-TAP-PARP10 or C-TAP-PARP10-G888W. Arrowheads indicate GST and the different GST-PARP10 fusion proteins. The labeled proteins, i.e. PARP10 full length and GST-PARP10(818-1025) are indicated by arrows. The experiment was performed twice with similar outcome.

In order to analyze which parts of the catalytic domain are essential for the trans-ADP- ribosylation by full-length TAP-PARP10, we prepared C-terminal deletion mutants of GST-PARP10(818-1025)-G888W. Deletion of the most C-terminal 30 aa of PARP10 completely abrogated the use of this mutant as substrate for the full-length enzyme (Fig. 21). Since this deletion mutant lacks the conserved ß-sheet 6 participating in the catalytic core domain (Fig. 17), it can be concluded that an intact catalytic core is essential for the trans-ADP-ribosylation by the full-length enzyme. This can be explained in two ways: either the binding of NAD+ by the “substrate” catalytic domain is Results and discussion 52 necessary to get ADP-ribosylated, because it might induce a conformational change, or the disintegration of the three-dimensional structure of the catalytic domain causes the loss of protein-protein interactions between the intact enzyme and the potential “substrate” catalytic domain. Both explanations underline the importance of structural integrity for the catalytic core motif. As expected, all analyzed C-terminal deletion mutants of the catalytically active GST-PARP10(818-1025) resulted in an abrogation of auto-ADP-ribosylation as well as trans-ADP-ribosylation of TAP-PARP10-G888W (Fig. 21). Regarding the importance of the deleted regions in the catalytic core, this is most likely due to the loss of its NAD+ binding capacity resulting in a dead enzyme.

Figure 21. The indicated PARP10 fragments were expressed as GST-fusion proteins and subjected to an enzymatic PARP assay with C-TAP-PARP10 or C-TAP-PARP10-G888W. Arrowheads indicate the different GST-PARP10 fusion proteins. A second experiment confirmed the results obtained from the displayed experiment.

ADP-ribosylation occurs on a number of distinct amino acids, including acidic residues, arginines, and cysteines, with distinct sensitivities of the respective chemical bond (Hassa et al., 2006). Auto-modified PARP10 was highly sensitive to hydroxylamine treatment, indicating an ester bond between ADP-ribose and acidic residues (Fig. 22A).

However, auto-modified PARP10 was insensitive to HgCl2 (Fig. 22B), which cleaves Cys-ADP-ribose linkages (McDonald and Moss, 1994). meta-Iodobenzylguanidine (MIBG), an inhibitor of arginine-specific mARTs (Smets et al., 1990), did not inhibit the Results and discussion 53 enzymatic activity of PARP10 (Fig. 22C). Thus cysteines or arginines were not modified by PARP10. mARTs can be inhibited specifically by Vitamin K1 (Banasik et al., 1992).

Figure 22. A. Immobilized GST-PARP10(818-1025) was auto-modified and then subjected to treatment with 1 M neutral NH2OH. Residual radioactivity was determined by autoradiography. B. Immobilized GST-PARP10(818-1025) was automodified with 32P-NAD+ and 50 M NAD+ at 30°C for 30 min. The modified proteins were then subjected to treatment with 1 mM neutral HgCl2. C. C-TAP-PARP10 was subjected to a PARP assay with the indicated concentrations of meta- Iodobenzylguanidine (MIBG), an inhibitor of arginine-specific mARTs. D. C-TAP-PARP10 was subjected to PARP assays with the indicated concentrations of different PARP inhibitors. Control experiments (0 M) were performed with the highest concentrations of solvent used for the inhibitors. IC50 values for PARP1 are indicated as derived from the literature (Banasik et al., 1992; Jagtap et al., 2005; Southan and Szabo, 2003; Woon and Threadgill, 2005). All experiments were at least performed twice with similar outcomes.

Results and discussion 54

In agreement with the conclusion drawn above that PARP10 is a non-classical mono- ADP-ribosyltransferase, Vitamin K1 did not interfere with PARP10 function (Fig. 22D). In addition the differential sensitivity to known PARP1 inhibitors distinguishes PARP10 from PARP1, despite the strong structural similarity (Fig. 22D). Regarding the small, but obvious differences in the catalytic center of bona fide PARPs like PARP1 and the novel PARPs functioning as mARTs, it is not surprising that especially many of the novel PARP inhibitors designed for PARP1 inhibition are inefficient in targeting PARP10 and presumably other novel PARPs. This also indicates that it should be possible to design potent inhibitors that are selective for the mARTs among the PARP family members. Such inhibitors might be useful tools to dissect the roles of mono- ADP-ribosylation and poly-ADP-ribosylation in cells.

One characteristic of the poly-ADP-ribosylation of PARP1 is its sensitivity to poly-ADP- ribose glycohydrolase (PARG) and ADP-ribose hydrolase 3 (ARH3), while ARH1 and ARH2 are inactive towards pADPr (Oka et al., 2006). ARH1 has been described to remove ADP-ribose from proteins modified on arginines (Takada et al., 1993). So far no enzymatic activity has been reported for ARH2. Therefore we tested whether PARG was capable of de-ADP-ribosylating C-TAP-PARP10. Indeed, we observed a more than 2-fold reduction of auto-modified PARP10 after PARG treatment (Fig. 23A). This suggested that PARG can remove the ADP-ribose from an acidic residue, in addition to hydrolyzing the glycosidic bond between ADP-ribose units. To test this further we used PARP1-E988K, a PARP1 mutant with low catalytic activity restricted to mono-ADP- ribosylation (Rolli et al., 1997). Importantly, this modification was reversed by PARG, supporting the notion that PARG has a mono-ADP-ribosyl-protein lyase activity; this activity has previously been ascribed to an enzyme distinct from PARG (Oka et al., 1984). It is important to note that previous studies obtained similar results, suggesting that PARG is able to remove mono-ADP-ribose from acidic residues (Desnoyers et al., 1995). To expand on this observation, auto-modified GST-PARP10(818-1025) was treated with PARG and ARH1-3. Both PARG and ARH3, but not ARH1 and 2, were able to de-ADP-ribosylate GST-PARP10(818-1025) (Fig. 23B).

Results and discussion 55

Figure 23. A. C-TAP-PARP10 and PARP1-E988K were immobilized on Protein A-Sepharose using specific antibodies, auto-modified and subsequently subjected to PARG treatment. Residual radioactivity was determined by autoradiography. B. Immobilized GST-PARP10(818-1025) was auto-modified and afterwards subjected to PARG/ARH assay using the indicated GST proteins. The displayed data are representative for at least two performed experiments.

Thus, in addition to their described pADPr glycohydrolase activity, PARG as well as ARH3 are likely able to remove the last ADP-ribose moiety from proteins ADP- ribosylated on acidic residues. Together these findings strengthen our conclusion that PARP10 mono-ADP-ribosylates acidic residues. Whether PARG and ARH3 indeed serve as de-ADP-ribosylating enzymes for PARP10-modified proteins in vivo has to be addressed in the future. This will require to develop reagents that are suitable for the analysis of proteins mono-ADP-ribosylated on acidic residues (see discussion below).

2.1.4 Identification of Glu882 as auto-ADP-ribosylation site It appears that PARPs modify acidic residues, but there is limited information on the identity of PARP-modified ADP-ribosylation sites; for example, the site(s) of auto- modification have not been mapped in PARP1, despite considerable effort (Hassa et al., 2006). To identify potential auto-modification sites in PARP10, we evaluated the Results and discussion 56 positions of acidic residues in PARP10 using our structural model. Mutation of four potentially accessible acidic residues at the extreme C-terminus, either alone or in combination, reduced the auto-modification only slightly (Fig. 24A), suggesting that the C-terminus is not efficiently ADP-ribosylated. We then turned to four surface exposed glutamates, E866, E870, E877, and E882, and mutated these residues individually to alanine. Of these mutants GST-PARP10(818-1025)-E882A was significantly less auto- ADP-ribosylated, while the other mutants showed no or less significant reduction (Fig. 24B). Similarly the GST-PARP10(818-1025)-E882Q was poorly auto-modified (data not shown). No reduction in enzymatic activity per se was observed for GST-PARP10(818- 1025)-E882A as determined by its ability to trans-ADP-ribosylate C-TAP-PARP10- G888W (Fig. 24C). Furthermore, the trans-ADP-ribosylation of the catalytically inactive GST-PARP10(818-1025)-G888W-E882A by the full-length C-TAP-PARP10 was also reduced (Fig. 24D). Thus these findings suggest that E882 is a major, but apparently not the only, acceptor site for PARP10 auto-ADP-ribosylation.

Although our in vitro data clearly show that E882A is a site targeted by the PARP10 auto-modification reaction, its importance in vivo and the functional relevance for the auto-modification remain elusive. Since antibodies recognizing mono-ADP-ribosylated acidic residues are commercially not available and mass spectrometric identification of ADP-ribosylation is technically not feasible due to the lability of the ester bond, it would be necessary to develop appropriate reagents to analyze the auto-modification of PARP10 and, even more important, the ADP-ribosylation of PARP10 substrates in vivo. The generation of antibodies targeting the PARP10 auto-modification site could be achieved by the enzymatic ADP-ribosylation of substrate peptides and the subsequent injection into rabbits. Polyclonal serum of the immunized animals should then be purified with the ADP-ribosylated peptides to get rid of the antibodies recognizing the unmodified peptide. Since ADP-ribose can be cleaved by pyrophosphatases, it will most likely be necessary to use a pyrophosphatase-resistant NAD+ analogue as described by Hilz and colleagues (Meyer and Hilz, 1986; Meyer et al., 1984).

Results and discussion 57

Figure 24. A. The indicated PARP10 fragments were expressed as GST-fusion proteins in the background of a catalytically inactive mutant (GST-PARP10(818-1025)-G888W) and incubated in the presence of C-TAP-PARP10 with 32P-NAD+ and 50 M NAD+ at 30°C for 30 min. The reaction products were analyzed by SDS-PAGE. B. The indicated GST-fusion proteins were subjected to a standard PARP assay. Incorporated radioactivity was detected and quantified on a phosphoimager. Relative ADP-ribosylation was calculated using mean values derived from four separate experiments. Depicted is a representative experiment. C and D. The indicated PARP10 fragments were expressed as GST-fusion proteins, auto- modified in the presence of C-TAP-PARP10-G888W (C) or C-TAP-PARP10 (D) and analyzed by autoradiography. All experiments were at least performed twice and showed results similar to the ones displayed.

To investigate the feasibility that E882 of PARP10 could serve as the auto-modification site, a model of the PARP10 auto-modification complex was constructed (Fig. 25A). In this model, a “substrate” PARP10 (S-PARP10) was complexed with an “enzymatic” PARP10-NAD+ complex (NAD-PARP10). The NAD+ was positioned based on a complex between diphtheria toxin and NAD+ (PDB ID 1TOX; (Bell and Eisenberg, 1996)). With relatively minor adjustments to loop regions, we were able to position the carboxylate of E882 in S-PARP10 very close to I987 of NAD-PARP10, such that the carboxylate of E882 occupies a position roughly equivalent to the position occupied by the carboxylate of the catalytic glutamate in PARP1 (Fig. 25A-C). Thus the pocket between the ribose-nicotinamide of NAD+ and I987 is sufficiently large to accommodate the glutamate of the substrate.

Results and discussion 58

Figure 25 (in collaboration with B.H. Shilton). A. Two views of the auto-modification complex consisting of a PARP10 molecule with bound NAD+ (surface representation, along with NAD+ as sticks with yellow carbon atoms) and a PARP10 substrate molecule (blue), including the side chain of E882, a site of ADP-ribosylation. B. Detail of the residues surrounding the E882 site of modification; residues from the substrate are illustrated with carbon atoms colored cyan with the sequence RPVEQV. C. The carboxylate of E882 (cyan carbons) occupies a position very close to that of the catalytic glutamate (E988) in PARP1 (magenta carbons) and can therefore act to stabilize the oxacarbenium ion transition state.

2.1.5 Substrate-assisted catalysis by PARP10 The ADP-ribosylation reaction has been well-studied, and a mechanism incorporating the most important features is illustrated in Figure 26A. NAD+ is bound by the enzyme, with the nicotinamide moiety positioned in a deep pocket and isolated from solvent; the lack of support for the positive charge on the nicotinamide promotes formation of the transition state (Berti et al., 1997). For the toxins, measurement of kinetic isotope effects indicate that the transition state bond orders are very small for both the leaving nicotinamide group and the incoming nucleophile and that the transition state has a high degree of oxocarbenium ion character (Berti et al., 1997; Parikh and Schramm, 2004; Rising and Schramm, 1997; Scheuring et al., 1998; Scheuring and Schramm, 1997). In other words, when the nucleophile has begun to form a bond with C1’, the Results and discussion 59 bond to the leaving group has almost been broken. Thus, for diphtheria toxin the mechanism has been termed a “dissociative” Sn2 reaction (Berti et al., 1997).

The catalytic glutamate does not appear to be important for binding of NAD+ since its mutation in PARP1 (Marsischky et al., 1995) as well as the bacterial toxins (Bohmer et al., 1996; Douglas and Collier, 1990) does not affect the dissociation constant for NAD+. Instead, the catalytic glutamate functions to stabilize the oxocarbenium ion transition state that develops during the reaction. On this basis, the carboxyl group of the catalytic glutamate must be very close to the C1’-N bond, and therefore well- positioned to stabilize the oxocarbenium ion transition state through a charge interaction.

Given this well-characterized and essential function of the catalytic glutamate, its replacement with isoleucine in PARP10 raises the question of how this enzyme can efficiently catalyze ADP-ribosylation. Both PARP1 and PARP10 catalyze ADP- ribosylation on substrate glutamate residues. PARP1 also has a polymerase activity and catalyzes elongation and branching reactions by adding ADP-ribosyl moieties to the initial glutamate-linked ADP-ribose and then to the growing pADPr chain. The nucleophile for the initial reaction is the carboxylate of the substrate glutamate, while the nucleophiles for the polymerization reactions are ribose hydroxyls. For PARP1, mutagenesis of the catalytic glutamate shows that these two reactions - chain initiation and polymerization - have different catalytic requirements. For example, mutation of the catalytic glutamate to alanine (E988A) abrogates polymerization, but the enzyme can still catalyze chain initiation, although at low rates compared to wild-type. Mutation of E988 to glutamine still results in loss of polymerization activity, but the mutant enzyme has only a slightly decreased ability to catalyze chain initiation. Finally, an E988D mutant catalyzes both chain initiation and elongation, but at reduced rates compared to wild-type (Marsischky et al., 1995; Ruf et al., 1998b). Thus, for PARP1 the degree of oxocarbenium ion stabilization by the residue at position 988 determines the catalytic properties of the enzyme. On this basis, the presence of isoleucine at the position of the catalytic glutamate in PARP10 could explain its lack of polymerase activity, and we reasoned that replacement of I987 with glutamate might restore a polymerase activity and improve its ability to catalyze chain initiation.

Results and discussion 60

Figure 26 (in collaboration with B.H. Shilton). A. The dissociative Sn2-type mechanism. In this mechanism, the transition state is stabilized by the catalytic glutamate and has a high degree of oxocarbenium ion character. B. The indicated GST-PARP10 fragments were incubated in the presence of C-TAP-PARP10- G888W with 32P-NAD+ and 50 M NAD+. The reaction products were analyzed by autoradiography. This experiment was performed twice with the same results. C. A fully dissociative substrate-assisted catalytic mechanism. The glutamate is attached to the substrate and is inserted into a position where it can promote the oxocarbenium ion transition state and react with it once it has formed.

To address whether replacing I987 by glutamate in PARP10 might be sufficient to obtain a polymerase, GST-PARP10(818-1025)-I987E was tested for catalytic activity. Unexpectedly, the I987E mutation made the protein almost completely inactive as a mono-ADP-ribosyltransferase (Fig. 26B). In other words, mutation of I987E not only failed to improve the activity of PARP10, but actually abrogated its auto-modification activity. In contrast, mutation of T999, which was suggested to correspond to the catalytic E988 of PARP1 (Ame et al., 2004), to glutamate had no effect on catalytic Results and discussion 61 activity (Fig. 26B). This is consistent with its location on the surface of our PARP10 model, away from the active site (data not shown). The loss of activity in the I987E mutant is difficult to explain given the conserved and virtually essential role for a catalytic glutamate in related ADP-ribosyltransferases, and also in light of the structural conservation of the enzyme, particularly in the active site region. Mutations at this position have been accommodated in many of the other related ADP- ribosyltransferases, and from the PARP10 structure there is no obvious reason why a glutamic acid could not be accommodated at position 987. On this basis, we concluded that PARP10 must use a different catalytic mechanism than the other ADP- ribosyltransferases, namely one that involves substrate-assisted catalysis.

For substrate-assisted catalysis to be operational in PARP10, the site of modification also fulfills the function of the catalytic glutamate, and must therefore occupy a position close to where the catalytic glutamate would normally reside. This is supported by our structural model that is described above (Fig. 25A-C). A substrate-assisted catalytic mechanism could work as follows. First, given the similarity in the structure of PARP10 with other ADP-ribosyltransferases, along with the identity of the NAD+ substrate, it is likely that the reaction proceeds through a similar oxocarbenium ion transition state. However, since PARP10 lacks a catalytic glutamate at position 987, the transition state is stabilized by a substrate glutamate and the reaction would proceed by a fully dissociative Sn1-type of mechanism (Fig. 26C). Insertion of the substrate glutamate into the region next to the nicotinamide ribose would promote formation of the oxocarbenium ion and cleavage of the ribose-nicotinamide bond, followed by nucleophilic attack on the oxocarbenium ion. A mechanism incorporating substrate- assisted catalysis explains the surprising loss of activity of the I987E mutant of PARP10: insertion of an acidic group in exactly the same region that the substrate glutamate must bind would prevent modification of the substrate.

Further experiments will be necessary to obtain additional data supporting the model of substrate-assisted catalysis by PARP10. These experiments can include the measurement of NAD+ glycohydrolase activity of GST-PARP10(818-1025) and the mutant GST-PARP10(818-1025)-I987E. Because the glycohydrolase activity is necessarily dependent on the catalytic glutamate (Bellocchi et al., 2006), it should be absent in PARP10-WT lacking the glutamate, but it should be restored in the PARP10- I987E mutant. Additional evidence for the proposed mechanism might be obtained by the determination of crystal structures of the catalytic PARP10 domain bound to the non-hydrolysable carba-NAD+ and by co-crystallisation with a potential substrate. Results and discussion 62

These structures will allow us to refine the structure of the active center at atomic resolution, to define the structural changes occurring through NAD+ binding and to identify the structural requirements for insertion of a substrate into the active center.

2.1.6 Functional relevance of the conserved loop region between ß-sheets 4/5 In addition to the amino acids in the catalytic center, the loop region differs between the three proposed PARP subfamilies (Fig. 17 and Table 1). The protein model suggests that for the substrate to have access to this region, the structure of the loop connecting ß-strands 4 and 5 is critical (Fig. 27A). In this regard, PARPs 6-8, 10-12, and 14-16 share a highly conserved feature: the loop for these proteins is very short. With the exception of PARPs 6 and 8, there is a conserved proline two residues N-terminal to the position normally occupied by the catalytic glutamate. Because of the limited rotation around the Phi bond, this proline residue places structural constraints on the polypeptide chain, and therefore the structure of the loop in the PARP12 structure is likely conserved in the other PARPs of this subfamily, including PARP10. Note that PARPs 6 and 8, which have an isoleucine in place of the catalytic glutamic acid, possess a short two residue turn between beta4 and beta5. Thus, the PARP enzymes of this subfamily most likely have a ß4-ß5 loop structure that allows insertion of a substrate-linked glutamate residue in place of the catalytic glutamate, which is missing from these enzymes. This structural arrangement is supported by our PARP10 dimeric model (Fig. 25A).

To address the relevance of the loop in PARP10 we generated several mutants in this region, including changing P985 to alanine. All these mutants had little or no catalytic activity (Fig. 27B). This was obvious for the activity towards the mutated protein itself as well as the catalytically inactive PARP10-G888W, which has an unaltered E882 and surrounding amino acids. These findings strongly indicate that indeed the loop is critical for positioning the catalytic/acceptor glutamate of the substrate in the catalytic center. The functional relevance of the ß4-ß5 loop that is suggested by the mutagenesis studies (Fig. 27B) could be supported by domain switch experiments in which the loop residues in PARP10 are replaced by loop residues from another mono-ADP- ribosyltranferase of the PARP family, e.g. PARP14. If the hypothesis that the loop plays an important role in substrate recognition as shown for the bacterial toxins (Han and Tainer, 2002; Sun et al., 2004) is correct, the resulting chimeric protein should loose its ability to modify full-length PARP10, but gain the ability to modify full-length PARP14.

Results and discussion 63

Figure 27 (in collaboration with B.H. Shilton). A. The PARP10 loop (gray) in comparison to the PARP1 loop (magenta) are shown relative to substrate PARP10 (cyan) with the indicated acceptor site E882. B. The indicated GST-PARP10 fragments with mutations in the loop region were subjected to a PARP assay with C-TAP-PARP10-G888W. The obtained results were confirmed by one further experiment with similar findings.

Employing substrate-assisted catalysis together with the loop structure would make PARP10 and related enzymes more specific for their substrates since the geometrical constraints required for modification are greater than they would be for the more conventional type of reaction seen for PARP1. Indeed PARP1 has a large number of substrates (Hassa et al., 2006). In contrast PARP10 is highly selective and several PARP1 substrates tested are not modified by PARP10 (Yu et al., 2005b). It might be possible to identify a potential consensus sequence for the PARP10 substrates by peptide scanning starting from a peptide that serves as PARP10 substrate, e.g. a peptide covering the region around the auto-modification site. The replacement of the surrounding amino acids might give indications which structural requirements and which charge of the amino acid side chains have to be given for efficient ADP- ribosylation by PARP10. Another approach to gain insights into the PARP10-mediated ADP-ribosylation and into its biological role would be a generic approach to identify PARP10 substrates. This might be achieved by using a proteome array that works analogous to a conventional DNA microarray. It contains several thousands of GST- fusion proteins spotted onto a nitrocellulose-coated glass carrier. The use of radioactively labeled NAD+ would allow us to identify the PARP10 substrates among the spotted proteins, and via positional mapping the labeled spots could be assigned to the corresponding proteins. This approach that is planned for the near future in our lab Results and discussion 64 will offer the possibility to screen for PARP10 substrates in a nearly proteome-wide fashion.

If the ß4-ß5 loop is really responsible for substrate recognition, one might expect a high grade of sequence variations in this region among the PARP family members. Indeed, a comparison of the loop sequences revealed no obviously conserved residues, but instead showed a great diversity (Otto et al., 2005). An inspection of the PARP10 loop sequence (CICQPS) showed the unique presence of two cysteine residues that are absent in the other novel PARPs 7 to 16. Since cysteine residues can serve as redox sensors (D'Autreaux and Toledano, 2007), we tested whether the addition of DTT or iodocetamide would influence the catalytic properties of PARP10 (Fig. 28). While DTT as reducing agent is able to reverse disulphide bonds resulting in free thiol groups, iodoacetamide reacts with free thiol groups leading to the formation of S- carboxyamidomethylcysteine, an irreversibly alkylated cysteine derivative. While DTT increased the catalytic activity of GST-PARP10(818-1025), no effect was observed for TAP-PARP10. In contrast, the addition of iodoacetamide to the reaction efficiently decreased the enzymatic activity of GST-PARP10(818-1025) as well as TAP-PARP10 in a concentration-dependent manner (Fig. 28). Thus, the effect of DTT might be restricted to the GST-fusion proteins and therefore DTT might act on the level of the GST tag, while the blockage of thiol groups by iodoacetamide is relevant for the catalytic activity of both the GST fusion protein as well as TAP-PARP10.

Figure 28. GST-PARP10(818-1025) and TAP-PARP10 were subjected to standard PARP assays with the indicated concentrations of DTT or iodoacetamide. The reaction mixture was separated by SDS- PAGE. The gel was stained with Coomassie blue, dryed and incorporated label was analyzed by autoradiography. Depicted are preliminary data from a single experiment. Results and discussion 65

Since potentially all cysteines in PARP10 are subject to alkylkation by iodoacetamide, it cannot be excluded that cysteines apart from the two residues in the PARP10 loop are responsible for the observed decrease in catalytic activity. Therefore, we analyzed GST-PARP10(818-1025)-C981S/C893S, in which both cysteines were replaced by serine residues. We observed in our PARP10 model of the catalytic domain that the loop cysteines would be able to form hydrogen bonds with residues in the active center and therefore might be relevant for catalysis (data not shown). This property should be conserved in the Cys/Ser mutants because the hydroxyl group should be able to form the same hydrogen bonds as the thiol group, but in contrast to the thiol group the hydroxyl group cannot be blocked by iodoacetamide. This led us to the prediction that GST-PARP10(818-1025)-WT and GST-PARP10(818-1025)-C981S/C893S should behave in the same way regarding their normal enzymatic activity, but iodoacetamide treatment would abrogate the activity in the wild-type but not in the mutant enzyme.

Figure 29. A. GST-PARP10(818-1025)-WT and –C981S/C983S were subjected to standard PARP assays with a constant amount of 32P-NAD+ and the indicated concentrations of ß-NAD+. The amount of incorporated label was determined by autoradiography of the electrophoretically separated reaction components. B, C. GST-PARP10(818-1025)-WT and –C981S/C983S were subjected to standard PARP assays with the indicated concentrations of DTT or iodoacetamide. The reaction mixture was separated by SDS-PAGE. The gel was stained with Coomassie blue, dryed and incorporated label was analyzed by autoradiography. Depicted are preliminary data from a single experiment.

As expected, no difference in the basal activity of GST-PARP10(818-1025)-WT and GST-PARP10(818-1025)-C981S/C893S was observed (Fig. 29A). Interestingly, their susceptibility to treatment with DTT and iodoacetamide was also comparable (Fig. 29B, C). Thus, it is likely that DTT indeed acts on the level of the GST tag and has no Results and discussion 66 specific effects on the two cysteine residues present in the ß4-ß5 loop of PARP10. Regarding the action of iodoacetamide, it might be possible that iodoacetamide lowers the pH in the enzymatic reaction thereby inhibiting the ADP-ribosyltransferase activity of PARP10. This suggestion is supported by the observation that addition of sample buffer to the iodoacetamide-treated reactions resulted in a switch from blue to yellow coloring indicating acidification of the sample. To obtain information about the iodoacetamide action on the cysteines in the ß4-ß5 loop of PARP10, it will be necessary in future experiments to exclude the unspecific pH decrease by iodoacetamide. This might be achieved by pre-incubation of the enzyme with iodoacetamide resulting in irreversible cysteine alkylation and subsequent removal of iodoacetamide prior to the enzymatic assay. In summary, the so far performed experiments allow no clear conclusion about the potential regulation of enzymatic PARP10 activity by the redox state of the cysteine residues in the ß4-ß5 loop.

2.1.7 Post-translational regulation of the catalytic PARP10 activity Our mass-spectrometric analysis of TAP-PARP10 revealed three phosphorylation sites in PARP10 targeted in vivo by so far unidentified kinases (data not shown). In order to investigate if these or other phosphorylation events contribute to the regulation of PARP10’s catalytic activity, we treated HA-PARP10 overexpressed in HeLa cells and immunoprecipitated by means of the HA tag with phosphatase and subjected it to a PARP assay. The effectiveness of the phosphatase treatment was controlled by the de-phosphorylation of the highly phosphorylated Rb protein in a parallel reaction (Fig. 30). The de-phosphorylation of TAP-PARP10 resulted in a loss of catalytic activity of approximately 50% suggesting a positive influence of the phosphorylations on catalytic PARP10 activity. Results and discussion 67

Figure 30. HA-PARP10 was overexpressed in HeLa cells, immunoprecipitated with a mAb targeted against the HA tag (3F10) and subjected first to a phosphatase assay and afterwards to a standard PARP assay. The phosphatase reaction was controlled on immunoprecipitated endogenous Rb protein which phosphorylation state was analyzed by a mixture of three different antibodies recognizing prominently phosphorylated residues in Rb. This result was confirmed by a second experiment with similar outcome.

A previous report analyzed the phosphorylation of PARP10 by Cyclin E/CDK2 and suggested that Cyclin E/CDK2 phosphorylates PARP10 on a single site, Thr-101 (Chou et al., 2006). The authors also observed a dependency of the catalytic activity on the presence of phosphorylated Thr-101 (Chou et al., 2006). This is contrary to the results obtained by our experiments showing that the catalytic domain per se, which does not contain Thr-101, is capable of auto- and trans-ADP-ribosylation. In addition, although phosphatase treatment resulted in reduced catalytic PARP10 activity, a robust basal activity was still present indicating that phosphorylations are not necessary for PARP10 to act as mono-ADP-ribosyltransferase, but rather have stimulatory effects. Since the kinase activity of Cyclin E/CDK2 is important for the transition from G1 to S phase (Harper and Adams, 2001) and the PARP10 interaction partner c-MYC is also involved in this transition, we decided to analyze if PARP10 serves as substrate for Cyclin E/CDK2 and which parts of PARP10 might contain the phosphorylation sites. In vitro kinase assays revealed a robust phosphorylation of TAP-PARP10 with recombinant, highly purified Cyclin E/CDK2 (Luscher-Firzlaff et al., 2006; Pandithage et al., 2008) (Fig. 31). GST-PARP10(1-255) served as a good substrate in our kinase assay, which is consistent with the finding that Thr-101 is a Cyclin E/CDK2 substrate and with Thr- 101 lying in a CDK consensus sequence (S/T-P-x-K/R). But obviously PARP10 Results and discussion 68 contains more than one Cyclin E/CDK2 phosphorylation site, because GST- PARP10(408-649) as well as GST-PARP10(600-868) were even better substrates than GST-PARP10(1-255) (Fig. 31). It is interesting to note that the catalytic domain was a poor substrate for Cyclin E/CDK2 suggesting that Cyclin E/CDK2 phosphorylation might either have no influence on the catalytic activity or that it might induce a conformational change in the full-length PARP10 with allosteric effects on the catalytic activity. Further experiments will be necessary to dissect the potential functions of the Cyclin E/CDK2-mediated phosphorylation of PARP10 and to identify the modification sites. Considering the fact that Cyclin E/CDK2 and c-MYC participate in the same regulatory cell cycle events, the functional importance of this post-translational modification of PARP10 should not be underestimated. It might be possible that this phosphorylation occurring in vivo most likely at the G1-S phase transition regulates the action of PARP10 on its interaction partner c-MYC. If the biological role of PARP10 in connection to c-MYC is unraveled, the possible regulatory function of Cyclin E/CDK2 should therefore be kept in mind.

Figure 31. TAP-PARP10-G888W and the GST fusion proteins of PARP10 indicated by arrowheads were subjected to a kinase assay with Cyclin E/CDK2. The reaction products were separated by SDS- PAGE and the incorporation of label was analyzed by autoradiography. Full-length PARP10 and the auto-modified GST-Cyclin E/CDK2 are marked by arrows. The results of a single in vitro kinase assay are displayed. Results and discussion 69

2.1.8 Biological significance of the PARP10-mediated ADP-ribosylation reaction Despite our extensive analysis of PARP10’s enzymatic in vitro properties, very little is known about the relevance of its activity in vivo. It was shown before that PARP10 is able to interfere with the transformation of REFs, but this ability was not dependent on catalytic activity (Yu et al., 2005b). Therefore, we decided to analyze a potential role of PARP10’s ADP-ribosyltransferase activity in the proliferation of cells by performing a colony formation assay in HeLa cells. This assay allows for assessing the proliferative capacity of transfected cells after selection with puromycin. The overexpression of PARP10 resulted in a clear reduction of methylene blue-stained cells suggesting an anti-proliferative effect of PARP10 in this setting (Fig. 32A). Importantly, an opposite effect was observed for the catalytically inactive PARP10-G888W suggesting a dependency on PARP10’s catalytic activity. The overexpression of p27 led to a very strong reduction in the number of stained cells correlating well with its role as inhibitor of the G1-S phase transition (Fig. 32B). In addition to the G888W mutant of PARP10, another catalytically inactive mutant, PARP10-H887E, also exhibited no anti- proliferative effect underlining the importance of an intact ADP-ribosyltransferase activity for the anti-proliferative phenotype observed for PARP10-WT (Fig. 32B).

Figure 32. A. HA-PARP10-WT-, HA-PARP10-GW-expressing plasmids and the corresponding backbone vector as control were transfected into HeLa cells along with the pBabePuro plasmid mediating puromycin resistance. After transfection, it was selected for transfected cells using puromycin. After several days of growth, colonies were stained with methylene blue. B. The experiment from A was repeated using an additional enzymatically inactive PARP10 mutant, PARP10-H887E, and p27, a known inhibitor of cell proliferation, as positive control.

Although the observed phenotype of PARP10 in the colony formation assay indicates an inhibitory role of PARP10 with respect to proliferation, it will be necessary to define the precise function of the PARP10-mediated ADP-ribosylation in vivo. The results Results and discussion 70 obtained from the above assay might result from an anti-proliferative or from a pro- apoptotic effect, both leading to the observed reduction in cell number. Thus, an examination of the cell cycle distribution of PARP10-overexpressing cells, e.g. by FACS analysis, might give a hint if PARP10 interferes with cell cycle progression. A more direct analysis of proliferation could be accomplished by a BrdU assay that measures the percentage of cells undergoing DNA synthesis during S-phase. Since cells have to overcome an important cell cycle check point before entering mitosis, a potential interference of PARP10 with mitotic entry could be analyzed by FACS analysis using an antibody recognizing the phosphorylation of histone H3 at Ser-10, a hallmark of mitotic cells (Prigent and Dimitrov, 2003). A reduced percentage of mitotic cells would suggest a role for the PARP10-mediated ADP-ribosylation in mitosis resulting in delayed cell cycle progression. Such a delay would cause a reduced cell number in the colony formation assay. If PARP10 would act as pro-apoptotic protein instead of interfering with cell cycle progression, the analysis of caspase activation, a staining of Annexin-V at the cell surface, the release of cytochrome-c from the mitochondria or the nuclear translocation of AIF would reveal an increase in the induction of apoptosis.

The phenotype of PARP10 in the colony formation assay is abrogated with the mutants PARP10-G888W and PARP10-H887E (Fig. 32). Since the two mutated residues participate directly in the NAD+ binding site, it is likely that the catalytic inactivity of these mutants is due to their inability to bind NAD+. Co-factor binding often triggers a conformational change in the catalytic center of enzymes potentially uncovering binding sites for interaction partners/substrates or modification sites. To exclude secondary effects due to the loss of NAD+ binding, the analysis of PARP10-I987E in the colony formation assay is desirable. This mutant most likely retains the ability to bind NAD+ but is unable to execute ADP-ribosylation due to interference of the active site glutamate with the inserted substrate glutamate (discussed above). From the in vitro results obtained with this mutant, it can be expected that I987E would behave like PARP10- G888W with respect to the proliferation of HeLa cells. In order to examine the so far unknown role of the auto-modification reaction, PARP10-E882A should be also used in the colony formation assay.

Since Chou et al. observed an accumulation of cells in G1-phase concomitant with a loss of cell viability after siRNA-mediated knockdown of PARP10 in HeLa cells (Chou et al., 2006), we generated pSuper-based shRNA vectors targeting different regions of the PARP10 mRNA within the ORF. We tested these constructs by co-expression Results and discussion 71 along with the PARP10-expressing plasmid pEVRF0-HA-PARP10 in HEK293 cells. The siRNAs siPARP10_1 and siPARP10_6 proved highly efficient in the knockdown of PARP10 protein, while a control siRNA targeting firefly luciferase exhibited no effect (Fig. 33A). Thus, we chose these siRNA constructs to examine the knockdown of PARP10 in the HeLa colony formation assay. Although the results from Chou et al. (Chou et al., 2006) suggested a growth-inhibitory effect, the siRNAs targeting PARP10 were indistinguishable from the control siRNA targeting luciferase (Fig. 33B). Based on our observations we conclude that PARP10 is dispensable for the proper growth of HeLa cells, but the level of the PARP10-mediated ADP-ribosylation has to be tightly controlled.

Figure 33. A. The efficiency of shRNA vectors targeting PARP10 was analyzed in HEK 293 cells. 5 g of pEVRF0-HA-PARP10 were co-transfected along with 15 g of the indicated shRNA vectors targeting Luciferase (siLuc_2) or PARP10 (siPARP10_1-6) into HEK293 cells. Three days after transfection, the cells were lysed in TAP lysis buffer and the levels of HA-PARP10 and Actin were analyzed by immunoblotting. The HA-PARP10 signal was corrected by the actin signal and normalized to the mock-transfected control. B. The effect of siRNA-mediated PARP10 knockdown on the proliferation of HeLa cells was assessed by colony formation assays as described in Fig. 32. The shown results were reproduced at least once in an independent experiment.

Although the PARP10-targeting siRNAs were tested on overexpressed protein, it will be necessary to demonstrate their efficiency at the endogenous level. Since the endogenous PARP10 is not detectable by a straight immunoblot of HeLa cell lysates, this might be achieved either by an IP-Western approach or by a quantitative RT-PCR analyzing the level of PARP10 mRNA. If the endogenous RNA is equally efficiently Results and discussion 72 targeted as the overexpressed mRNA, we would expect efficient knockdown of the low abundant endogenous protein. Thus the above drawn conclusion should hold true that PARP10 is not necessary for HeLa cell proliferation.

2.2 PARP10’s subcellular localization and nucleocytoplasmic shuttling

2.2.1 PARP10 fragments display a heterogeneous subcellular localization As shown previously, the mainly cytoplasmic PARP10 is subject to nucleocytoplasmic shuttling and accumulates in the nucleus after treatment with Leptomycin B (LMB), a potent inhibitor of the Crm1-mediated nuclear export (Yu et al., 2005b). This is due to the presence of a functional NES in the C-terminal half of the protein whose mutation resembles the effect of LMB treatment (Yu et al., 2005b). While the nuclear export seems to be mainly dependent on this NES, it is unclear how PARP10 enters the nucleus. Bioinformatical searches for a classical nuclear localization signal were not successful suggesting that PARP10 might be imported into the nucleus via an alternative mechanism. In order to analyze which regions in PARP10 determine its subcellular localization we created CFP/YFP-tagged fusion proteins of overlapping PARP10 fragments (Fig. 34). All fusion proteins were expressed to an equal extent and comparable to the CFP/YFP tag alone (Fig. 34). Only minor breakdown products, if any, were observed after overexpression in COS-7 cells, and cleavage of the fusion proteins resulting in a free CFP/YFP tag was observed only in a low percentage (Fig. 34).

The CFP/YFP tag alone possesses a molecular weight of about 60 kDa (Fig. 34). Although its size is at the upper limit for passive diffusion through the nuclear pores (Allen et al., 2000; Terry et al., 2007), it is still able to cross the nuclear envelope passively resulting in a more or less equal distribution between nucleus and cytoplasm (Fig. 36). An additional increase in size by fusion to a heterologous protein should most likely abrogate the passive diffusion rendering the transport of the CFP/YFP fusion proteins across the nuclear envelope dependent on localization signals allowing facilitated diffusion with the help of karyopherins. Since all analyzed CFP/YFP fusion proteins possess apparent molecular weights of more than 90 kDa, it is very unlikely that these chimeric proteins are subject to passive diffusion across the nuclear membrane. Thus an accumulation of these fusion proteins in the nucleus should be indicative for ongoing nuclear import mechanisms. Results and discussion 73

Figure 34. The upper panel shows the overlapping fragments covering the whole protein chosen for expression as fusion proteins. The overexpression of the CFP/YFP-tagged fusion proteins and the expression of the tag alone was determined by an immunoblot of COS-7 lysates using an anti-GFP antibody.

The analysis of the subcellular localization of the created CFP/YFP fusion proteins revealed a heterogeneous pattern with some fusion proteins present exclusively in the cytoplasm and others present in the cytoplasm and the nucleus (Fig. 35). As expected, the localization of PARP10(408-649) harboring the NES was restricted to the cytoplasm (Fig. 35). PARP10(1-255) and PARP10(600-868) also showed a mainly cytoplasmic localization, although a minor nuclear fraction was clearly detectable (Fig. 35). Regarding aa 600-868 it is worth to consider that most of the NES covering aa 598-607 is still present in this fragment and might be sufficient to mediate Crm1- dependent export of this fragment, although the export might be impaired compared with PARP10(408-649). PARP10(206-459) covering the Glycine-rich region in PARP10 was equally distributed between cytoplasm and nucleus indicating that the aa 206-459 of PARP10 harbor sequences that enable the CFP/YFP fusion protein to enter the nucleus (Fig. 35). The uniform distribution suggests that neither an efficient NES mediates nuclear export nor a classical NLS forces the chimeric protein into the nucleus. PARP10(818-1025) representing the catalytic domain exhibited a slight accumulation in the nucleus suggesting that an active mechanism drives the nuclear Results and discussion 74 import of this fragment (Fig. 35). Interestingly, the catalytically inactive PARP10(818- 1025)-GW showed a more pronounced nuclear accumulation (Fig. 35). Since the absence of catalytic activity favored the nuclear localization of PARP10(818-1025), the auto-ADP-ribosylation or the ADP-ribosylation of substrates might have an impact on the nuclear import or the retention in the nucleus. Conceivable scenarios would be that the ADP-ribosylation interferes with the nuclear import by modifying proteins essential for the import process or that ADP-ribosylation results in the release from nuclear proteins holding up PARP10(818-1025) in the nucleus.

Figure 35. For explanations, see top of next page. Results and discussion 75

The indicated CFP/YFP-fusion proteins were overexpressed in COS-7 cells and their subcellular distribution analyzed in PFA-fixed cells after Hoechst staining of nuclear DNA. Shown are merged pictures of CFP/YFP and Hoechst (left panel) and the corresponding brightfield images (middle panel) at 200x magnification. In addition, a representative confocal image with a red DRAQ5 DNA counterstain is displayed, while fusion proteins appear in blue (right panel). The displayed pictures are representative of several independent experiments showing very similar subcellular distributions.

In summary, the localization of the analyzed PARP10 fragments suggests that at least one functional NES in PARP10 drives its nuclear export and that at least two regions in PARP10 might contribute to the entry of the full-length protein into the nucleus, with PARP10(818-1025) being the best candidate for possessing sequences that mediate nuclear uptake.

Because the above experiments were all performed in the presence of ongoing nuclear export processes, one might miss an existing NLS if its effect is masked by a stronger NES. Thus we decided to analyze the steady-state localization of the CFP/YFP- PARP10 fusion proteins in the presence of LMB. As expected, the LMB treatment had no effect on the CFP/YFP tag alone but inhibited the nuclear export of PARP10(408- 649) disrupting the function of the characterized NES and resulting in a more or less equal distribution between nucleus and cytoplasm (Fig. 36). LMB did not interfere with the subcellular localization of PARP10(206-459), (818-1025) and (818-1025)-GW indicating that these regions of PARP10 do not contain a classical NES which correlates with their significant nuclear fraction observed in the steady-state localization analysis (Fig. 36). The cytoplasmic localization of PARP10(1-255) was also not altered by the LMB-induced blockage of the Crm1-dependent export suggesting that this fragment might be exported from the nucleus via an alternative export mechanism or that it might be not able to enter the nucleus (Fig. 36). Interestingly, the cytoplasmic localization of PARP10(600-868) showed no susceptibility to LMB indicating that the absence of a significant nuclear fraction of this fusion protein is not due to the presence of the only partially deleted NES but due to the lack of nuclear import (Fig. 36).

Results and discussion 76

Results and discussion 77

Figure 36. The indicated CFP/YFP-fusion proteins were overexpressed in COS-7 cells and treated for 3.5h with 20 nM Leptomycin B (LMB) or a methanol solvent control prior to fixation with PFA. Their subcellular distribution was analyzed after Hoechst staining of nuclear DNA. Shown are merged pictures of CFP/YFP and Hoechst at 200x magnification. The displayed pictures are representative of several independent experiments showing very similar subcellular distributions.

In order to confirm the function of the previously described NES in the context of the CFP/YFP fusion proteins, we mutated the functional NES in PARP10(408-649). In addition, we mutated the partially deleted NES in PARP10(600-868) to rule out any effect on the subcellular localization of the fusion protein. As expected, mutation of the NES resulted in nuclear accumulation of PARP10(408-649) confirming the results obtained with LMB (Fig. 37). Additional treatment of CFP/YFP-PARP10(408-649)- dNES with LMB did not lead to a more pronounced localization in the nucleus indicating that no further Crm1-dependent export mechanisms mediate the nuclear export of this fragment. CFP/YFP-PARP10(600-868)-dNES showed a cytoplasmic localization comparable to the one observed for the corresponding wild-type fusion protein (Fig. 37). This result supports our hypothesis that the cytoplasmic localization of this chimeric protein is not dependent on the partially present NES but is most likely due to the lack of nuclear import. Consistent with our previous observations with the wild-type protein, the NES-mutated version was not sensitive to treatment with Leptomycin B ruling out the presence of a classical NES (Fig. 37).

Figure 37. The indicated CFP/YFP-fusion proteins were processed as explained in Fig. 36.

Results and discussion 78

In summary, the steady-state analysis of the CFP/YFP-PARP10 fusion proteins in the absence or presence of Crm1-dependent nuclear export revealed that the previously identified NES from aa 598-607 is the only functional NES in PARP10. In addition, our studies show that no classical NLS exists because one of the typical features of an NLS, the efficient targeting of chimeric proteins to the nucleus resulting in an almost completely nuclear localization (Lange et al., 2007), was observed for none of the analyzed fusion proteins. Since it is possible that an NLS is not encoded in the primary sequence but in the tertiary structure of a protein (Sessler and Noy, 2005) and thus might be missed in our analysis of PARP10 fragments, it is important to analyze the full-length protein in a similar setting (described below). Although no classical NLS was found by our studies, we provide some evidence that sequences within aa 206-649 and aa 818-1025 of PARP10 participate in the nuclear import of PARP10. Since PARP10(206-459) and PARP10(408-649) share identical amino acid sequences in their overlapping region, it is tempting to speculate that a potential import-mediating region is localized within aa 408-459.

The nuclear import of PARP10 might be via a piggyback mechanism along with so far unidentified, NLS-containing interaction partners or it might be mediated by the direct interaction with importins. Further work is necessary to distinguish these two scenarios, which might be achieved e.g. by analyzing the potential interaction of the imported PARP10 fragments with known importins. To confirm that the observed nuclear import of the analyzed fusion proteins is indeed dependent on actively driven import mechanisms, it would be important to analyze their nuclear import in the absence of facilitated diffusion. A simple experiment would be to cool down the cells to 4°C, which would inhibit the active import mechanism but not significantly impair passive diffusion (Plafker and Macara, 2000). It is important to note here that the C-terminal part of PARP10 interacts with the oncoprotein c-MYC, a nuclear protein possessing a strong NLS (Henriksson and Luscher, 1996; Luscher and Eisenman, 1990). Since c-MYC would be a potential candidate for targeting PARP10(818-1025) to the nucleus, it would be interesting to analyze the localization of the corresponding CFP/YFP fusion protein in the absence of c-MYC.

2.2.2 The nucleocytoplasmic shuttling of PARP10 fragments Since the analysis of the steady-state localization of the PARP10 fragments provides only static information about the subcellular localization, we chose the iFLAP approach to measure the dynamics of nucleocytoplasmic shuttling processes. iFLAP (inverted Fluorescence Localization After Photobleaching) makes use of the selective YFP Results and discussion 79 bleaching of CFP/YFP fusion proteins to follow up the movement of the bleached molecules within the cell. Because the CFP and YFP are inseparably coupled to the analyzed protein, CFP can serve as an internal control and the ratio of CFP and YFP fluorescence provides information about where the bleached molecules have moved. A cell adjacent to the bleached cell serves as control for potential artifacts caused by photobleaching during the image acquisition. Since we were interested in nucleocytoplasmic shuttling, we chose an experimental setting with bleaching in the cytoplasm. If the CFP/YFP ratio in the nucleus changes in this setting, this will be due to an exchange of molecules between the cytoplasm and the nuclear compartment resulting in the uptake of bleached molecules from the cytoplasm. It is important to note here that due to intrinsic technical limitations, namely the barely detectable fluorescence in the nucleus, it is difficult to analyze almost exclusively cytoplasmic proteins with this approach and care has to be taken with the interpretation of the obtained results. Therefore we carefully analyzed the raw data from the displayed experiments to exclude any artificial results. We came to the conclusion that these experiments, although difficult, especially the ones with PARP10(408-649), were technically sound and therefore interpretable without major limitations.

We observed a high mobility of all analyzed CFP/YFP fusion proteins within the cytoplasm indicated by the rapid distribution of the bleached molecules from the bleach ROI to the other cytoplasmic areas (Fig. 38). An inspection of their nucleocytoplasmic shuttling behavior revealed a striking difference between PARP10(408-649) and all other PARP10 fragments (Fig. 38). While most fragments showed little exchange of bleached molecules between the cytoplasm and the nucleus visualized by the blue- or greenish-colored nucleoplasm, the CFP/YFP-PARP10(408-649) molecules were rapidly exchanged between both compartments indicated by the observation that the nuclear envelope seemed to present only a negligible barrier for the movement of these molecules and that the nucleoplasm was rapidly filled with cytoplasmically bleached molecules visualized by the red coloring (Fig. 38). Importantly, disruption of the NES in PARP10(408-649) led to a significant decrease in the nucleocytoplasmic shuttling resulting in levels slightly higher than for PARP10(206-459) (Fig. 38). This is most likely due to the abrogation of nuclear export reducing the rate of molecular exchange between cytoplasmic and nuclear fusion proteins. The iFLAP analysis of PARP10(818-1025)-WT and -GW revealed a complete absence of any exchange between nucleus and cytoplasm within the time frame of the experiment (Fig 38). At the first glance this appears to be contradictory to its steady-state localization because the nuclear fraction of this PARP10 fragment has to enter the nucleus from the cytoplasm. Results and discussion 80

Figure 38. The indicated CFP/YFP fusion proteins were expressed in COS-7 cells and subjected to an iFLAP approach using a confocal LSM510 microscope (Zeiss, Germany) as described in the experimental procedures. A pre-bleach confocal image is displayed for every experiment (left panel). Pseudo-colored images of different time points illustrate the movement of bleached molecules. Blue represents an equal ratio of CFP and YFP fluorescence intensity and red indicates the detection of only CFP fluorescence resulting from bleaching of the YFP molecules. The bleach ROI is marked by white boxes. The depicted experiments are representative of at least four independent experiments per analyzed PARP10 fragment.

Results and discussion 81

A possible explanation might be that the nuclear import occurs very slowly and thus cannot be visualized during the 10 minutes of the experiment. A slow import would be in accordance with the high levels of cytoplasmic PARP10(818-1025) in the steady- state localization indicating a potential oversaturation of the import mechanism. The same might be true for PARP10(206-459) that also showed a low level of nucleocytoplasmic transport (Fig. 38). In summary, our iFLAP experiments revealed that only PARP10(408-649) exhibits a rapid nucleocytoplasmic shuttling that is dependent on its functional NES while none of the other regions in PARP10 tend to shuttle across the nuclear envelope in a similar rapid fashion. This is to a large degree in accordance with our steady-state localization analysis demonstrating that PARP10(408-649) is able to enter the nucleus and that it is actively exported by means of its NES (Figs. 36 and 37). Thus the central part of PARP10 might mainly contribute to its subcellular localization. In order to clarify the role of this region in context of the full-length protein, it might be interesting to analyze a CFP/YFP-PARP10 fusion protein in which the aa 408-649 were removed.

2.2.3 The nucleocytoplasmic shuttling of full-length PARP10 So far we restricted our analysis to overlapping fragments of PARP10. In order to analyze the subcellular localization and the nucleocytoplasmic shuttling of the full- length protein, we expressed CFP/YFP-PARP10 in COS-7 cells. Immunoblot analysis showed that the chimeric protein was expressed as a single band with an appararent molecular weight of more than 200 kDa (Fig. 39A). Surprisingly, an inspection of its subcellular localization revealed a remarkable cytoplasmic staining with several bright foci, which will be discussed and followed up in detail below (Fig. 39B). As expected, CFP/YFP-PARP10 was localized exclusively in the cytoplasm due to the presence of its NES as seen previously (Yu et al., 2005b).

Results and discussion 82

Figure 39. A. CFP/YFP-PARP10 overexpression in COS-7 cells was determined by an immunoblot of COS-7 lysates using an anti-GFP antibody. B. Its subcellular distribution was analyzed in PFA-fixed cells after Hoechst staining of nuclear DNA. Shown are merged pictures of CFP/YFP and Hoechst (left panel) and the corresponding brightfield images (middle panel) at 200x magnification. In addition, a representative confocal image with a DRAQ5 DNA counterstain is displayed (right panel).

In order to characterize the NES function in the context of the full-length protein, we analyzed the subcellular localization of CFP/YFP-PARP10-WT and -PARP10-dNES in the absence or presence of LMB. In contrast to the results obtained by Yu et al. (Yu et al., 2005b) with the HA-tagged protein, we did not observe a considerable accumulation of PARP10 in the nucleus after LMB treatment. Instead the inhibition of Crm1-dependent nuclear export resulted in a minor nuclear accumulation of CFP/YFP- PARP10 in only some cells, with most cells still showing an almost exclusive cytoplasmic localization (Fig. 40). The same is true for CFP/YFP-PARP10-dNES that was also not sensitive to LMB treatment (Fig. 40).

Results and discussion 83

Figure 40. The indicated CFP/YFP-fusion proteins were overexpressed in COS-7 cells and treated for 3.5h with 20 nM Leptomycin B (LMB) or a methanol solvent control prior to fixation with PFA. Their subcellular distribution was analyzed after Hoechst staining of nuclear DNA. Shown are merged pictures of CFP/YFP and Hoechst at 200x magnification.

The obvious contrast between the previous results (Yu et al., 2005b) and the ones from the present study cannot be explained by cell type specific differences because we obtained similar results in U2OS cells used by Yu et al. and in HeLa cells (data not shown). It cannot be excluded that the kind of the tag makes the difference in the two experimental settings. While the results with HA-PARP10 suggest an efficient nuclear import of the fusion protein (Yu et al., 2005b), the present results with CFP/YFP- PARP10 indicate that the fusion protein is not efficiently targeted to the nucleus but instead hardly enters the nucleus and only in a low percentage of cells (Fig. 40). If the used CFP/YFP tag potentially interferes with nuclear import e.g. by masking import- mediating sequences, has to be clarified in future experiments using HA-PARP10 or other tagged PARP10 versions. A further explanation might be that the localization of HA-PARP10 was determined by indirect immunocytochemistry after fixation and permeabilization of cells (Yu et al., 2005b), which might lead to artifacts. The localization of CFP/YFP-PARP10-WT and -dNES was analyzed in living as well as in fixed cells without any changes due to fixation (own observations). Thus we can exclude an artifactual localization of the fusion proteins in the present study. It has to be further mentioned that the conclusions drawn from Yu et al. (Yu et al., 2005b) are based on the analysis of a very limited number of cells providing no information about the appearance of the whole cell population. In contrast, the overview images of CFP/YFP-PARP10 (Fig. 40) allow the assessment of the subcellular localization of a representative population of transfected cells. Although our data provide some evidence that full-length PARP10 enters the nucleus inefficiently, it is important to note that nuclear fractions of PARP10 exist at least in some cells (Fig. 40). If the appearance of nuclear PARP10 is somehow regulated e.g. in a cell cycle dependent fashion has to be clarified by further experiments. Importantly, experiments using Results and discussion 84 bimolecular fluorescence complementation (Hu et al., 2002) clearly demonstrate an interaction of PARP10 and its interaction partner c-MYC in the nucleus (unpublished results from J. Lüscher-Firzlaff) supporting the existence of a nuclear PARP10 fraction, which might be physiologically relevant in terms of regulating c-MYC function.

In order to obtain more information about the nucleocytoplasmic shuttling of full-length PARP10, we subjected CFP/YFP-PARP10-WT and -dNES to the iFLAP approach. Both fusion proteins diffused freely in the cytoplasm as seen for the PARP10 fragments (Fig. 41). In accordance with our steady-state analysis of full-length PARP10 we were not able to measure significant nucleocytoplasmic shuttling of PARP10-WT or PARP10-dNES within the experimental time frame (Fig. 41). Although the mutation of the NES resulted in the appearance of a nuclear PARP10 fraction (Figs. 40 and 41B), the iFLAP analysis revealed only low levels of shuttling across the nuclear envelope (Fig. 41B). This result is comparable with the ones for PARP10(206-459) and PARP10(818-1025) indicating that the nuclear import occurs very slowly.

Figure 41. The indicated CFP/YFP fusion proteins were expressed in COS-7 cells and subjected to an iFLAP approach using a confocal LSM510 microscope (Zeiss, Germany) as described in the experimental procedures. A pre-bleach confocal image is displayed for every experiment (left panel). Pseudo-colored images of different time points illustrate the movement of bleached molecules. Blue represents an equal ratio of CFP and YFP fluorescence intensity and red indicates the detection of only CFP fluorescence resulting from bleaching of the YFP molecules. The bleach ROI is marked by white boxes.

The analysis of CFP/YFP-PARP10 with the iFLAP approach was technically complicated due to the hardly detectable nuclear fluorescence rendering the analysis prone to misinterpretations. It has to be mentioned that the result displayed in Fig. 41A is by far the best result obtained for CFP/YFP-PARP10 within a set of six experiments and that most of the other measurements were not analyzable due to signal artifacts created by the calculation procedure. Thus care has to be taken by the interpretation of these data. Results and discussion 85

The iFLAP result for CFP/YFP-PARP10-WT suggests that no nucleocytoplasmic shuttling is detectable within 10 minutes (Fig. 41A). This is somehow surprising considering the rapid shuttling of PARP10(408-649) (Fig. 38C) because the full-length protein should in theory possess all the features of the analyzed fragments, namely the functional NES around aa 600 and the two potential import-mediating regions from aa 206-649 and aa 818-1025. Speculating about the reasons for this discrepancy, it might be due to the large difference in the molecular weight of CFP/YFP-PARP10 and CFP/YFP-PARP10(408-649) because the import of larger cargoes occurs significantly slower than that of smaller ones (Ribbeck and Gorlich, 2002). Since the crystal structure of PARP10 has not been resolved yet, it also cannot be excluded that the regions mediating nuclear import in PARP10(408-649) are not surface-exposed in full- length PARP10 and therefore not accessible for interaction with potential NLS- containing binding partners and/or importins. A less likely possibility could be that an additional NES is present in the full-length protein that is reconstituted from amino acids of separated regions of the protein. Since LMB treatment did not cause strong nuclear accumulation of CFP/YFP-PARP10 this potential NES had to act independent of Crm1. As outlined above the iFLAP analysis of CFP/YFP-PARP10 proved very difficult, and a strong interpretation is impossible with the data obtained so far. This is underlined by the observation that CFP/YFP-PARP10-dNES underwent a more rapid shuttling than PARP10-WT (Fig. 41B). Since a functional NES significantly contributes to the exchange of molecules between the cytoplasm and the nucleus, one would expect a decrease in nucleocytoplasmic shuttling if it is mutated, as seen for PARP10(408-649) (Fig. 38). Thus a conclusive answer to the emerged questions can only be given through further experiments including the resolution of the PARP10 crystal structure, the screening for potential NLS-containing binding partners or interacting karyopherins and careful analysis of the endogenous protein. Unfortunately, the levels of endogenous PARP10 are very low in most adherent cell lines well-suited for microscopic analysis, while PARP10 is considerably higher expressed in suspension cell lines originating from the hematopoietic system. Thus the analysis of endogenous PARP10 in e.g. U937 cells requires the use of a cytospin apparatus in order to place the cells onto a cover slip and the establishment of a staining procedure with appropriate controls to verify that indeed endogenous PARP10 is stained. Results and discussion 86

2.2.4 PARP10 enriches in dynamic cytoplasmic foci As indicated above, for full-length PARP10 we unexpectedly observed a prominent enrichment in cytoplasmic foci (Figs. 39 and 40). Since we suspected that the CFP/YFP-tag might target PARP10 for aggregation, we repeated the analysis with HA- PARP10 and PARP10-DsRed resulting again in the formation of prominent foci (data not shown). Although at present we cannot exclude the possibility that these foci are aggregation-driven artifacts, we made several observations that argue against simple aggregation. First, while a classical aggresome is a single perinuclear irregular shaped structure (Spector, 2006), the observed PARP10 foci are small round structures and are present in numbers from 0-30 (Figs. 39, 40, 42, and data not shown). Second, titrations of the plasmid encoding CFP/YFP-PARP10 did neither influence the amount nor the size of PARP10 foci, which would be expected if these structures would develop upon over-expression (data not shown). Third, preliminary experiments showed that co-expression of the chaperone Hsp70, which reduces the formation of aggregates (Gilks et al., 2004), had no effect on the formation of PARP10 foci (data not shown). To gain further insights into the formation and behavior of these cytoplasmic foci in a physiological setting, we decided to perform time-lapse microcopy in living cells. Analysis of COS-7 cells transfected with CFP/YFP-PARP10 by confocal microscopy revealed that the PARP10 foci are highly dynamic (Fig. 42). While at the beginning of the experiment several foci per cell were visible, these foci moved within the cytoplasm and tend to fuse during the time course of the experiment ending up with fewer and larger foci after about 4 hours of observation (Fig. 42, compare time points 0 and 220 min). Interestingly, one of the observed cells most likely entered mitosis indicated by breakdown of the nuclear envelope and rounding of the cell accompanied by the loss of the PARP10 foci (Fig. 42, time point 160 min). Since the time-lapse microscopy was performed on a confocal microscope, it cannot be doubtlessly concluded that the foci indeed have disintegrated. Instead it might be possible that due to the rounding of the cell the PARP10 foci have moved out of focus and thus were not visible any longer.

Results and discussion 87

Figure 42. The movement of CFP/YFP-PARP10 was followed up by confocal live-cell imaging using a LSM510 microscope (Zeiss, Germany). The displayed images were selected from the indicated time points. Note that the foci tend to fuse and that only very few foci occur de novo. The cell in the center of the group enters mitosis by 160 min.

In order to resolve the question if PARP10 foci vanish during mitosis, we switched to a conventional fluorescence microscope for the time-lapse microscopy and extended the observation time up to more than 20 hours. In addition, we decided to analyze the PARP10 foci in a cell line of human origin and transfected HeLa cells with CFP/YFP- PARP10. We observed again the movement of PARP10 foci in the cytoplasm and their tendency to fuse (Fig. 43). Importantly, in one cell we were able to follow up the disintegration of two larger foci and the subsequent formation of several smaller foci (Fig. 43, white box). This observation clearly indicates that PARP10 foci are dynamic, can disintegrate and can form de novo arguing against aggregation artifacts driven by over-expression. The vanishing of the two large foci was temporally associated with a rounding of the cell (Fig. 43, time point 900 min). Although this might be indicative for a beginning of mitosis, it is important to note that the cell neither underwent mitotic division nor re-settled within the next six hours of observation. In contrast, two non- transfected cells clearly passed through mitosis within a normal time frame as observed in the brightfield images (Fig. 43, parental cells and sister cells marked by yellow asterisks). An additional cell expressing CFP/YFP-PARP10 also rounded up early in the experiment and did not re-settle within the observation period (Fig. 43, marked by yellow hash mark), and none of the other transfected cells underwent normal mitosis, although the doubling time of HeLa cells is within the range of the time frame of the time-lapse microscopy. Results and discussion 88

Figure 43 (in collaboration with A.S. Sechi). CFP/YFP-PARP10 expressing HeLa cells were analyzed by epifluorescent live-cell imaging for more than 20h. Fluorescence and brightfield images from the indicated time points are displayed. Please note that the cells expressing CFP/YFP-PARP10 do not undergo cell division during the time period, while some non-transfected cells do (marked by yellow asterisks). A CFP/YFP-PARP10 expressing cell rounded up during the imaging and did not re-settle even after several hours (marked by yellow hash mark). The fusion of PARP10 foci and subsequent de novo formation of several smaller foci can be followed up in the cell marked by the white box.

Based on these preliminary data one might speculate that the CFP/YFP-PARP10- expressing cells might exhibit a disturbed cell cycle progression, potentially caused by a reduced ability to enter mitosis and/or a delay in mitotic progression. Such cell cycle malfunction would be a possible explanation for the growth-inhibitory effect observed in the HeLa cell colony formation assays (Fig. 32). Further time-lapse microscopy analyses will be necessary to get a better idea about the behavior of PARP10 foci throughout the cell cycle. An especially useful tool for this kind of experiments would be the use of genetically engineered cells whose cell cycle stages can be visualized in vivo by fluorescence techniques (Sakaue-Sawano et al., 2008).

Results and discussion 89

2.2.5 The N-terminal domains in PARP10 participate in foci formation Since the formation of cytoplasmic PARP10 foci was only observed in the full-length protein but not in the CFP/YFP-PARP10 fragments (Figs. 35 and 39), we decided to generate N-terminal deletions of CFP/YFP-PARP10 lacking the potential RNA/DNA recognition motifs to evaluate their potential implication in foci formation. In addition, the appearance of PARP10 foci resembles the one of RNA-processing particles, P bodies and stress granules (Spector, 2006), indicating a potential role of PARP10’s RNA recognition motif and its Glycine-rich region. Indeed, the deletion of its N-terminal regions severely compromised the formation of cytoplasmic PARP10 foci (Fig. 44, upper panels). Although the N-terminal parts of PARP10 were important for the development of the PARP10-enriched cytoplasmic bodies, they were not essential as indicated by the observation that most of the cells transfected with CFP/YFP- PARP10(257-1025) and -PARP10(552-1025) still displayed foci although at a lower frequency (Fig. 44). Accordingly, the analysis of COS-7 cells transfected with CFP/YFP-PARP10(1-459) revealed that the aa 1-459 are not sufficient for the formation of the foci, which is also true for the central part of PARP10 from aa 408-868 (Fig. 44, lower panels). Thus the development of PARP10 foci seems to be dependent on its C-terminal catalytic domain, although the catalytic domain itself is not sufficient (Figs. 35 and 44). The inspection of the subcellular localization of PARP10(1-459) also supported our suggestion that sequences within aa 208-459 mediate nuclear import. While CFP/YFP-PARP10(1-255) was localized mainly in the cytoplasm due to the lack of nuclear import (Figs. 35 and 36), the addition of further C-terminal regions up to aa 459 rendered the fusion protein able to enter the nucleus as indicated by the equal distribution between cytoplasm and nucleus (Fig. 44).

Results and discussion 90

Figure 44. The indicated CFP/YFP fusion proteins were overexpressed in COS-7 cells and their subcellular distribution was analyzed in PFA-fixed cells after Hoechst staining of nuclear DNA. Shown are merged pictures of CFP/YFP and Hoechst (left panel) and the corresponding brightfield images (middle panel) at 200x magnification. In addition, a representative confocal image with a red DRAQ5 DNA counterstain and the fusion proteins in blue is displayed (right panel).

In order to estimate the influence of the N-terminal regions on the PARP10 foci formation, we counted the number of foci per cell in the populations of transfected cells expressing CFP/YFP-PARP10-WT, -PARP10(257-1025) and -PARP10(552-1025). This quantitative approach revealed striking differences between the cells expressing full-length PARP10 or the N-terminally deleted versions (Fig. 45A). Regarding their capacity to develop PARP10 foci, the deletion of the RRM dramatically increased the percentage of cells without foci compared to PARP10-WT. Further deletion of the Glycine-rich region did not result in major changes (Fig. 45B). The differences between the populations of cells expressing PARP10-WT or PARP10-dN mutants were statistically highly significant as proved by a 2 test (Fig. 45B). The N-terminal deletions also significantly affected the number of foci observed per cell, with only very few cells with more than 5 foci being detected (Fig. 45C). Again no significant differences between the PARP10-dN1 (aa-257-1025) and the PARP10-dN2 (aa 552-1025) population were observed (Fig. 45C). Thus the N-terminal RRM contributes to the formation of PARP10-enriched cytoplasmic foci by increasing the percentage of cells Results and discussion 91 possessing foci in general and by increasing the percentage of cells containing numerous foci in particular.

Figure 45. The foci formation by CFP/YFP-PARP10-WT, -dN1 (aa257-1025) and -dN2 (aa552-1025) was analyzed after overexpression in COS-7 cells. A. The populations of WT-expressing (n=63), dN1-expressing (n=48) and dN2-expressing cells (n=39) were subdivided into cells with no foci, with 1-5 foci or with more than 5 foci and displayed as pie diagram with the percentage of each sub-population indicated. B. The populations were sub-divided into cells with or without foci and displayed as column diagram. A statistical 2 test was performed to verify that the PARP10-dN1 and -dN2 populations are different from the PARP10-WT population in terms of foci formation. The test showed a high significance with p<0.005. C. The foci-forming cells of each population were subdivided into cells with 1-5 foci or more than 5 foci. Note that abrogation of the N-terminal protein domains nearly abolishes the occurrence of cells with more than 5 foci.

Although we have not yet analyzed if the RRM in PARP10 is indeed functional in terms of RNA binding, we consider it highly likely from its implications on the development of PARP10 foci. Since RNA recognition motifs are often found in proteins mediating RNA transport or processing (Farina and Singer, 2002; Lunde et al., 2007; Maris et al., 2005) and PARP10 foci resemble the appearance of RNA-processing particles, a contribution of PARP10’s RRM in the foci formation would make sense. Future experiments have to clarify if the formation of PARP10 foci could be influenced e.g. by modulating the levels of cytoplasmic RNA or stalling the translation of mRNAs. Results and discussion 92

2.2.6 PARP10 foci seem to represent novel cytoplasmic bodies At first glance, the cytoplasmic PARP10 foci resemble the appearance of RNA- processing particles, but also a vesicular or endosomal origin cannot be excluded. Therefore we analyzed the co-localization of PARP10 with different markers of cytoplasmic sub-structures. As a control for the occurrence of PARP10 in the foci of CFP/YFP-PARP10-transfected cells, we stained PARP10 with a specific antibody. The complete overlap of both stainings confirmed the presence of PARP10 in the cytoplasmic foci (Fig. 46A). Co-localization experiments with PARP10 and the endosomal markers EEA1 and Rab5 revealed no significant overlap between both substructures indicating that PARP10 foci do not represent endosomal structures (Figs. 46B and C). PARP10 foci did also not co-localize with the Golgi compartment (Fig. 46D). Since we could demonstrate that the RRM is involved in the formation of PARP10 foci (Figs. 44 and 45) and the foci are similar to P bodies and stress granules (Anderson and Kedersha, 2008; Eulalio et al., 2007; Parker and Sheth, 2007; Spector, 2006), we analyzed if PARP10 might be a component of P bodies or stress granules. Therefore we co-expressed CFP/YFP-PARP10 and RFP-tagged versions of the stress granule component TIA-1 and the P body component Dcp1a. Although especially the TIA-1-induced cytoplasmic foci exhibited a similar appearance like the PARP10 foci, we observed no co-localization of PARP10 and TIA-1 or Dcp1a (Figs. 46E and F). From these experiments we concluded that the cytoplasmic PARP10 foci are neither related to endosomes nor show co-localization with RNA-processing particles, at least in a setting where markers for RNA-processing particles were over-expressed along with CFP/YFP-PARP10. Results and discussion 93

Figure 46. The co-localization of PARP10 foci with markers of different cytoplasmic structures was analyzed in PFA-fixed COS-7 cells by immunocytochemistry. A. The presence of CFP/YFP-PARP10 in the observed foci was verified with an anti-PARP10 antibody (5H11) and a secondary anti-rat-Cy3 antibody. B. Endosomes were stained by an anti-EEA1 antibody and a secondary Cy3-coupled antibody. C. Rab5 was co-expressed and analyzed with an anti-Rab5 antibody and a secondary Alexa555- coupled antibody. D. The co-localization of DsRed-PARP10 and the Golgi apparatus was analyzed by an anti- GM310 antibody and a secondary Cy2-coupled antibody. E. CFP/YFP-PARP10 and RFP-TIA1 were co-expressed and their localization analyzed in PFA-fixed COS-7 cells. F. CFP/YFP-PARP10 and RFP-Dcp1a were co-expressed and their localization analyzed in PFA-fixed COS-7 cells. Results and discussion 94

2.2.7 PARP10 foci might be related to RNA-processing particles Despite the obvious lack of co-localization between PARP10 foci and the overexpressed components of RNA-processing particles, we extended our co-localization studies of PARP10 and P bodies or stress granules. In collaboration with Georg Stöcklin, a well-known expert in the field of RNA-processing particles (Gilks et al., 2004; Kedersha et al., 2005; Stoecklin and Anderson, 2007; Yamasaki et al., 2007), we were able to analyze a potential connection between PARP10 and these sites of RNA decay and storage in more detail. The staining of endogenous P bodies through their core component Hedls (Fenger-Gron et al., 2005; Yu et al., 2005a) confirmed our previous result that PARP10 foci and P bodies are distinct cytoplasmic substructures (Fig. 47A). The same is true for PARP10 foci and stress granules that were induced by arsenite or FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) treatment and stained by their component HuR (Gallouzi et al., 2000). Thus the cytoplasmic PARP10- enriched bodies can be clearly separated from RNA-processing particles based on their localization.

Figure 47. The co-localization of CFP/YFP-PARP10 and RNA-processing particles was analyzed in COS- 7 cells (in collaboration with G. Stöcklin, DKFZ Heidelberg). For explanations, see top of next page. Results and discussion 95

A. CFP/YFP-PARP10 was overexpressed in COS-7 cells and endogenous Hedls present in P- bodies (PBs) was stained with an anti-S7K antibody and a red-fluorescent secondary antibody. A merge picture with an additional DAPI stain of the nuclear DNA is shown to assess a potential co-localization. B. CFP/YFP-PARP10 was overexpressed in COS-7 cells and endogenous HuR present in stress granules (SGs) was stained with an anti-HuR antibody and a red-fluorescent secondary antibody. The formation of stress granules was stimulated by treatment with arsenite or FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). A merge picture with an additional DAPI stain of the nuclear DNA is shown to assess a potential co-localization.

Since stress granules are not present in unstressed cells but form under stress conditions where translation of mRNAs is stalled (Anderson and Kedersha, 2008), we decided to analyze if the induction of stress granule promoting stress would have an influence on the localization of PARP10 foci with respect to components of RNA- processing particles. Therefore we co-expressed CFP/YFP-PARP10 and the P body components RFP-Dcp1a or RFP-Lsm1 and analyzed their localization in the absence or presence of stress (Fig. 48). In support of our previous analysis, we observed no co- localization of PARP10 foci and Dcp1a in untreated cells (Fig. 48A). Surprisingly, treatment of the cells with arsenite or FCCP resulted in an at least partial, sometimes complete co-localization of PARP10 and Dcp1a in cytoplasmic foci (Figs. 48B and C). Analysis of the co-localization between PARP10 and Lsm1 revealed a significant overlap even in untreated cells, which was significantly enhanced by stress treatment (Figs. 48D-F).

Based on these observations we concluded that although PARP10 foci and P bodies are distinct from each other in unstressed cells, the induction of stress that inhibits translation of mRNAs and causes the formation of stress granules results in a significant co-localization between P body components and PARP10 in the cytoplasmic PARP10 foci. Interestingly, overexpressed RFP-Dcp1a co-localizes well with endogenous P bodies, while RFP-Lsm1 does not (G. Stöcklin, personal communication). This implies that under stress conditions PARP10 foci might not only co-localize with Dcp1a but also with other P body components. The overlap between CFP/YFP-PARP10 and RFP-Lsm1 in unstressed cells suggest that Lsm1 might be a component of P bodies but might also be recruited to PARP10 foci.

Results and discussion 96

Figure 48. The co-localization of CFP/YFP-PARP10 and components of RNA-processing particles was analyzed in COS-7 cells (in collaboration with G. Stöcklin, DKFZ Heidelberg). A. CFP/YFP-PARP10 and RFP-Dcp1a was co-expressed in COS-7 cells and their distribution analyzed in fixed cells. A merge picture with an additional DAPI stain of the nuclear DNA is shown to assess a potential co-localization. B,C. The co-localization of PARP10 and Dcp1a was analyzed after treatment with arsenite or FCCP. D. CFP/YFP-PARP10 and RFP-Lsm1 was co-expressed in COS-7 cells and their distribution analyzed in fixed cells. A merge picture with an additional DAPI stain of the nuclear DNA is shown to assess a potential co-localization. E, F. The co-localization of PARP10 and Lsm1 was analyzed after treatment with arsenite or FCCP.

The functional connection between PARP10 and P body components has to be analyzed in further detail e.g. by performing interaction assays between PARP10 and these proteins. Since Dcp1a as well as Lsm1 are proteins involved in the decapping of RNA (Parker and Sheth, 2007), it will be interesting to test if PARP10 also plays a role in the processing of RNA which might be among others decapping, deadenylation or degradation of RNA. Little is known about the regulatory processes which occur within P bodies and stress granules and which determine the assembly or disintegration of these structures. Obviously, these functional processes have to be tightly regulated due to the general implications on the cellular translation. Since PARP10 possesses Results and discussion 97 mono-ADP-ribosyltransferase activity thereby mediating a reversible and potentially highly dynamic post-translational modification, it would be important to analyze if potential substrates for PARP10 exist in RNA-processing particles and if the modification of these substrates plays a regulatory role for P body function. Conclusions 98

3. Conclusions

Here we report that one of the novel PARP family members, PARP10, lacks polymerase activity while showing robust ADP-ribosyltransferase activity. PARP10 also lacks a catalytic glutamate that has a critical function in all of the previously characterized ADP-ribosyltransferases. We show that PARP10 like PARP1 modifies acidic residues and that the auto-ADP-ribosylation reaction occurs in trans. Molecular modeling revealed that the catalytic glutamate is replaced by an isoleucine in PARP10 and that no residue in the active center can functionally substitute for it. Strikingly, a PARP10-I987E mutant, in which the catalytic glutamate is restored, is completely devoid of activity. On this basis, we propose that PARP10 employs a mechanism of substrate-assisted catalysis (SAC) (Dall'Acqua and Carter, 2000; Kosloff and Selinger, 2001), where the glutamate that is modified fulfills the function of the catalytic glutamate. Such a mechanism explains why PARP10 does not function as a polymerase.

Since PARP14, an additional novel PARP enzyme that lacks the catalytic glutamate, possesses enzymatic activity comparable to PARP10, i.e. ADP-ribosyltransferase activity but no detectable polymer formation, our findings suggest that PARP10 is the prototypical enzyme of a subclass of the PARP family. Thus while PARPs 1-5 function as polymerases, the novel PARPs 6-16 are mono-ADP-ribosyltransferases. These lack the conserved catalytic glutamate and we suggest that they all may use substrate- assisted catalysis for ADP-ribosylating substrate. There are two exceptions within this PARP subfamily, PARPs 9 and 13, which lack the conserved histidine in the catalytic center in addition to the glutamate. The histidine is required for NAD+-binding. Indeed these two PARPs do not possess auto-ADP-ribosylation capacity. In summary we propose to subdivide the PARP family into three subfamilies, the first with bona fide pADPr polymerase activity (PARPs 1-5), the second with mono-ADP-ribosyltransferase activity (PARPs 6-8, 10-12, 14-16), and the third without catalytic activity (PARPs 9 and 13).

Intracellular mono-ADP-ribosylation has been postulated as a mechanism to regulate many different aspect of cell physiology. However the only enzymes described to mono-ADP-ribosylate substrates in cells are different bacterial toxins, while the mammalian mono-ADP-ribosyltransferases (ecto-enzymes) appear to be localized strictly extracellularly (Aktories and Barbieri, 2005; Corda and Di Girolamo, 2003; Hassa et al., 2006; Holbourn et al., 2006). Intracellular mono-ADP-ribosylation may Conclusions 99 result from the action of PARG on substrates carrying pADPr or by inefficient catalysis of polymerases. However there is no evidence to support either model. Finally Sirtuins have been suggested to function as mono-ADP-ribosyltransferases, in particular SIRT4 and SIRT6 (Haigis et al., 2006; Liszt et al., 2005). Since the NAD+ binding motif and the mode of catalysis is distinct between Sirtuins and PARP enzymes (Otto et al., 2005; Ruf et al., 1998b; Sauve et al., 2006), it is unlikely that ADP-ribosyltransferases of the Sirtuin family target acidic residues and employ substrate-assisted catalysis. Therefore our discovery of a mono-ADP-ribosylating class of PARP enzymes represents the first account of intracellular enzymes mono-ADP-ribosylating acidic residues. Importantly, the overexpression of PARP10 impairs the growth of HeLa cells dependent on mono- ADP-ribosyltransferase activity suggesting an important role for the PARP10-mediated ADP-ribosylation reaction in physiological processes. The mechanism of substrate- assisted catalysis proposed here, which uses a substrate glutamate and possibly an aspartate for catalysis, together with eight additional PARP family members potentially able to mono-ADP-ribosylate, suggests that mono-ADP-ribosylation of acidic residues might be a wide spread post-translational mechanism to control protein function. In addition mono-ADP-ribosylation might serve as acceptor sites for poly-ADP-ribose polymerases, possibly expanding the functional range of PARPs 1-5 considerably. It will now be important to identify the substrates that are mono-ADP-ribosylated by PARP10 and other PARP-like mono-ADP-ribosyltransferases to elucidate the physiological role of this modification.

As described previously, PARP10 shuttles between the nucleus and the cytoplasm (Yu et al., 2005b). Here we present data confirming that PARP10 is able to enter the nucleus upon inhibition of Crm1-mediated nuclear export. PARP10 contains a single functional NES mediating its export and being responsible for its mainly cytoplasmic localization. Despite careful analysis we were not able to define a classical NLS in PARP10, but we propose that three regions within PARP10 exhibit NLS-like features enabling nuclear entry. Future experiments will have to clarify if all of these regions contribute to the nucleocytoplasmic shuttling of PARP10 and if nuclear uptake is mediated by direct interaction with importins or via a piggyback mechanism through interaction with an NLS-containing protein.

Interestingly, PARP10 enriches in prominent cytoplasmic foci. These are dynamic, tend to fuse and can form de novo as confirmed by time-lapse microscopy. PARP foci likely do not represent previously described cytoplasmic substructures. Importantly, the deletion of the RNA recognition motif in PARP10 significantly reduces the formation of Conclusions 100 these foci indicating an important role for RNA in the subcellular localization of PARP10. Although the capacity of the RRM to bind RNA has not been analyzed yet, the influence on the development of PARP10 foci suggests that the RRM exerts a physiological role. We demonstrate that PARP10 foci are distinct from RNA-processing particles, i.e. P bodies and stress granules, but that some core components involved in RNA decay are shared between both cytoplasmic substructures, which is significantly enhanced under stress conditions that stall translation of mRNAs. Thus PARP10 might play a role in the processing of RNA and in cellular stress responses.

Future work will be directed towards understanding the SAC-based ADP-ribosylation reaction in more detail and towards the identification and analysis of PARP10 substrates and interaction partners to gain insights into its biological function. In addition, it will be necessary to develop novel tools for the analysis of mono-ADP- ribosylated proteins. The analysis of the subcellular localization of endogenous PARP10 will help to elucidate the relevance of the observed PARP10 foci, and further experiments have to be performed in order to understand the physiological role of PARP10 in these foci, which might be linked to RNA processes.

Experimental Procedures 101

4. Experimental Procedures

4.1 Consumables and Reagents Consumables Consumables were purchased from: Amersham Biosciences, Ansell, Becton Dickinson, Beranek, Biometra, Bio-Rad, Brand, Braun, Corning, Costar, Eppendorf, Falcon, Fisher Scientific, Fuji, Greiner, Hartenstein, Integra Biosciences, Kimberley-Clark, Merck, Millipore, Nalgene, Nerbe Plus, Nunc, Roth, Sarstedt, Sartorius, Schleicher&Schuell, Stratagene, Terumo, TPP, VWR, WillCo Wells.

Reagents Reagents met at least the criteria for the purity standard p.a. and were purchased from: Amersham Biosciences, AppliChem, Baker, Bayer, BD Biosciences, Biozym, Calbiochem, Clontech, Delta, Difco, Fluka, GE Healthcare, Gibco, Invitrogen, Invivogen, KMF, Fermentas, Merck, MP Biomedicals, New England Biolabs, Perkin Elmer, Qiagen Riedel-de-Haën, Roche, Rockland, Roth, Sigma, Stratagene, USB, Zymo Research.

4.2 Oligonucleotides The following oligonucleotides were purchased from MWG Biotech, Germany:

Oligo name sequence 5’ 3’ siPARP10#1_1 GAT CCC CGG GTA GAG GGA TTA TGA CAT TCA AGA GAT GTC ATA ATC CCT CTA CCC TTT TTG GAA A siPARP10#1_2 AGC TTT TCC AAA AAG GGT AGA GGG ATT ATG ACA TCT CTT GAA TGT CAT AAT CCC TCT ACC CGG G siPARP10#2_1 GAT CCC CGT GCA GGG ACT GTG ACA ATT TCA AGA GAA TTG TCA CAG TCC CTG CAC TTT TTG GAA A siPARP10#2_2 AGC TTT TCC AAA AAG TGC AGG GAC TGT GAC AAT TCT CTT GAA ATT GTC ACA GTC CCT GCA CGG G siPARP10#3_1 GAT CCC CCT TGA AGG ACC GGA TAT GAT TCA AGA GAT CAT ATC CGG TCC TTC AAG TTT TTG GAA A siPARP10#3_2 AGC TTT TCC AAA AAC TTG AAG GAC CGG ATA TGA TCT CTT GAA TCA TAT CCG GTC CTT CAA GGG G siP10#4for GAT CCC CTG GGT CCC ATG GAG ATC ACT TCA AGA GAG TGA TCT CCA TGG GAC CCA TTT TTG GAA A siP10#4rev AGC TTT TCC AAA AAT GGG TCC CAT GGA GAT CAC TCT CTT GAA GTG ATC TCC ATG GGA CCC AGG G siP10#5for GAT CCC CAG TGG CAG AAC GAG TGT TGT TCA AGA GAC AAC ACT CGT TCT GCC ACT TTT TTG GAA A siP10#5rev AGC TTT TCC AAA AAA GTG GCA GAA CGA GTG TTG TCT CTT GAA CAA CAC TCG TTC TGC CAC TGG G siP10#6for GAT CCC CAG GCC TTG AAG AGG TGG ACT TCA AGA GAG TCC ACC TCT TCA AGG CCT TTT TTG GAA A siP10#6rev AGC TTT TCC AAA AAA GGC CTT GAA GAG GTG GAC TCT CTT GAA GTC CAC CTC TTC AAG GCC TGG G Experimental Procedures 102 attB1-PARP10-5 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAA CAA CCT GGA GCG TCT GGC attB2-PARP10 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA AGT GTC TGG GGA GCG GC PARP10delC1rev GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA CTG GGT GTC GTG GAA GAT GAC G PARP10delC2rev GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA GTA GCG CAG GAG CAC GTG GCC AGG AC PARP10delC3rev GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA GTA GCG GTC CTG CAC CGA CAG GGA G PARP10delC4rev GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA TGC CGT CGT GCC GTG GTA CAG CAC C attB1-PARP10C30 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CGC GCT GCC CAC CCA CCT CAT CAC attB1-PARP10C50 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CGA CAG CGC CGT GGA CTG CAT C attB1-PARP7 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT CCA AGT CCC TGT TTC TGC AG attB1-P7aa332 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CTC CAC ACC ACC CTC TAG CAA TG attB2-PARP7 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA AAT GGA AAC AGT GTT ACT GAC TTC attB1-PARP9 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CGA GGT CCT TAT GGC TGC CTT TC attB2-PARP9 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA ATC AAC AGG GCT GCC ACT TGC G attB1-PARP12cat GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT GCA GAA GCA GAA TGG AGG GAA GG attB1-P12aa283 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAG CTT TCA AGA TAA GTG CCA TAG AGT TC attB2-PARP12 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA CTG TCG GCT GCT GAA CAG GGA G attB1-PARP13 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CCA GGA GGA CTT TTG CTT CCT ATC attB1-P13aa604 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA CTG CCT AAA TGT GAT AAG ATA CTC AG attB2-PARP13 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA ACT AAT CAC GCA GGC TTT GTC TTC attB1-mPARP14 GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CGA TTT CAC GGT GGA CTT GAG C attB2-mPARP14 GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TTA CTG CCT AAA TGT GAT AAG ATA CTC AG PARP10G888Wfor GCC GGT GCC GTC GTC CAG TGG TAC AGC ACC TGC PARP10G888Wrev GCA GGT GCT GTA CCA CTG GAC GAC GGC ACC GGC PARP10E1005A_for CCA CCT CAT CAC CTG CGC GCA CGT GCC CCG CGC TTC CC PARP10E1005A_rev GGG AAG CGC GGG GCA CGT GCG CGC AGG TGA TGA GGT GG D1013D1014A_for CGC TTC CCC CGC CGC CCC CTC TGG GC D1024A_for GGC CGC TCC CCA GCC ACT TAA GAC CCA G I987E_for GCA TCT GCC AGC CCA GCG AGT TCG TCA TCT TCC ACG A I987E_rev TCG TGG AAG ATG ACG AAC TCG CTG GGC TGG CAG ATG C P10T182E_for ACC CAG GCG CTG CCC GAG CAC CTC ATC ACC TGC P10T182E_rev GCA GGT GAT GAG GTG CTC GGG CAG CGC CTG GGT Experimental Procedures 103

P10R192K_for GCG AGC ACG TGC CCA AGG CTT CCC CCG ACG A P10R204K_for CTC TGG GCT CCC GGG CAA GTC CCC AGA CAC TTA AG E866Afor GCA GCA GCA GTA TGC GCT GTA CCG GGA GC E866Arev GCT CCC GGT ACA GCG CAT ACT GCT GCT GC E870Afor GCT GTA CCG GGC GCG CCT GCT GC E870Arev GCA GCA GGC GCG CCC GGT ACA GC E877Afor GCA GCG ATG CGC GCG GCG CCC GG E877Arev CCG GGC GCC GCG CGC ATC GCT GC E882Afor GCG CCC GGT GGC GCA GGT GCT GT E882Arev ACA GCA CCT GCG CCA CCG GGC GC E882Qfor GGC GCC CGG TGC AGC AGG TGC TG E882Qrev CAG CAC CTG CTG CAC CGG GCG CC del981-984neu_for CTA CGA CAG CGC CGT GGA CCC CAG CAT CTT CGT CAT CT del981-984neu_rev AGA TGA CGA AGA TGC TGG GGT CCA CGG CGC TGT CGT AG del983neu_for CCG TGG ACT GCA TCC AGC CCA GCA TCT TC del983neu_rev GAA GAT GCT GGG CTG GAT GCA GTC CAC GG P985Afor ACT GCA TCT GCC AGG CCA GCA TCT TCG TC P985Arev GAC GAA GAT GCT GGC CTG GCA GAT GCA GT D980AI982Sfor CAG CGC CGT GGC CTG CAG CTG CCA GCC CA D980AI982Srev TGG GCT GGC AGC TGC AGG CCA CGG CGC TG D980AI982SP985Afor GCG CCG TGG CCT GCA GCT GCC AGG CCA GCA TCT D908AI982SP985Arev AGA TGC TGG CCT GGC AGC TGC AGG CCA CGG CGC P10C981SC983Sfor GCG CCG TGG ACA GCA TCA GCC AGC CCA GC P10C981SC983Srev GCT GGG CTG GCT GAT GCT GTC CAC GGC GC

4.3 Plasmids Eukaryotic expression plasmids pBabe Puro This is a retroviral mammalian expression vector driving the expression of a puromycin resistance gene under the control of a SV40 early promoter (J.P. Morgenstern, and H. Land (1990) Nucleic Acids Research 18(12): 3587-96). pCMV-Rab5 This vector drives the expression of human Rab5 under control of a CMV promoter (A. Krüttgen). pCS2+-p27 A cDNA encoding human p27 was cloned into the EcoRI site of pCS2+ to drive expression of p27 under control of a CMV promoter (J. Vervoorts). pEQ176P2 This vector was routinely used for control transfections for CMV-containing vectors if the respective backbone vector for the transfected plasmid was not available. It is derived from pEQ176 which drives expression of ß-galactosidase under control of a CMV promoter. Most of the cDNA encoding ß- galactosidase was cut out by a PvuII restriction digest (J. Lüscher-Firzlaff). pEVRF0-HA This vectors allows for the expression of N-terminally HA- tagged proteins in eukaryotic cells under control of a strong CMV promoter (Matthias et al., 1989). The vector contains pSP65 plasmid sequences, a human CMV promoter/enhancer, the translation initiation region from the HSV thymidine kinase, the splicing and polyadenylation signals from the rabbit ß-globin gene and the SV40 origin of replication. Experimental Procedures 104

pEVRF0-HA- The full-length cDNA encoding the 1025 aa PARP10 protein PARP10 was ligated into pEVRF0-HA from pSport-p150 T+B using the KpnI and XbaI restriction sites (S. Schreek). pEVRF0-HA- This vector was obtained from pEVRF0-HA-PARP10 by site- PARP10-G888W directed mutagenesis using the oligos PARP10G888Wfor and PARP10G888Wrev (E. Poreba). pEVRF0-HA- This vector was obtained from pEVRF0-HA-PARP10 by site- PARP10-H987E directed mutagenesis using appropriate oligos (E. Poreba, J. Lüscher-Firzlaff). pEVRF0-HA- The SphI/BssHII fragment from pBS-PARP10-dNES was PARP10-dNES subcloned into the plasmid pEVRF0-HA-PARP10 using the same restriction sites (M. Yu). pECYFP This vector drives the expression of a eCFP/YFP-double tag under control of a CMV promoter. A Kanamycin/Neomycin resistance gene is included for selection in bacteria and mammalian cells, respectively (B. Giese, AG Müller-Newen). pECYFP-PARP10 PARP10 cDNA was cut out of GW-pD-PARP10 with Bsp1407I and filled with Klenow enzyme and ligated into pECYFP cut with Asp718I and filled with Klenow enzyme (K. Montzka). The reading frame was corrected by digesting the plasmid DNA with HindIII, filling up with Klenow enzyme and re-ligation (H. Schuchlautz). pECYFP-PARP10- PARP10-dNES cDNA was cut out of GW-pD-PARP10-dNES dNES with Bsp1407I and filled with Klenow enzyme and ligated into pECYFP cut with Asp718I and filled with Klenow enzyme (K. Montzka). The reading frame was corrected by digesting the plasmid DNA with HindIII, filling up with Klenow enzyme and re- ligation (H. Schuchlautz). pDsRed-Monomer- This vector allows for the expression of a monomeric mutant N1 from the tetrameric Discosoma sp. red fluorescent protein. The expression is driven by a CMV promoter. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed- Monomer (Clontech). pDsRed-Monomer- This vector allows for the expression of a C-terminally DsRed- N1-PARP10 tagged PARP10. cDNA encoding PARP10 was obtained from pcDNA5/FRT/TO/C-TAP-PARP10 by restriction digest with BamHI, subsequent mung bean nuclease treatment and HindIII digestion of plasmid DNA. pDsRed-Monomer-N1 was prepared in the same way. After gelextraction vector DNA and insert DNA were ligated (H. Schuchlautz). pDsRed-Monomer- This vector allows for the expression of a C-terminally DsRed- N1-PARP10-G888W tagged PARP10. cDNA encoding PARP10 was obtained from pcDNA5/FRT/TO/C-TAP-PARP10-G888W by restriction digest with BamHI, subsequent mung bean nuclease treatment and HindIII digestion of plasmid DNA. pDsRed-Monomer-N1 was prepared in the same way. After gelextraction vector DNA and insert DNA were ligated (H. Schuchlautz). GW-pHA This vector is derived from pEVRF0-HA which was made compatible with the Gateway system by insertion of the Gateway cassette Reading frame C1 (RfC1) into the SmaI site (R. Lilischkis). Experimental Procedures 105

GW-pECYFP The vector pECYFP was made compatible with the Gateway system by insertion of the Gateway cassette Reading frame C1 (RfC1) into the SmaI site. It allows for the expression of N- terminally CFP/YFP-tagged proteins (K. Montzka). GW-pECYFP- These vectors were created by a LR reaction with GW-pECYFP PARP10 (1-255), and the corresponding pDONR/Zeo entry vectors (H. (206-459), (408- Schuchlautz). 649), (600-868), (818-1025), (818- 1025) GW GW-pECYFP- These vectors were created by a LR reaction with GW-pECYFP PARP10 (257-1025), and the corresponding pDONR/Zeo entry vectors (L. Milke). (552-1025), (1-459), (408-868) pRFP-Dcp1a This vector drives the expression of RFP-tagged human Dcp1a (G. Stöcklin). pRFP-Lsm1 This vector drives the expression of RFP-tagged human Lsm1 (G. Stöcklin). pRFP-TIA-1 This vector drives the expression of RFP-tagged human TIA-1 (G. Stöcklin). pSuper-siLuc#2 This vector drives the expression of a functional shRNA targeted against the luciferase mRNA from the histone H1 PolIII promoter (C. Cornelissen). pSuper- These vectors were derived from pSuper by insertion of the siPARP10#1, 2, 3, 4, hybridized oligos siPARP10#1, 2, 3, 4, 5, 6 between the BglII 5, 6 and HindIII sites. They drive the expression of shRNAs designed for targeting the PARP10 mRNA (C. Cornelissen, H. Schuchlautz).

Prokaryotic expression plasmids GW-pGST This vector is derived from pGEX-4T-2 (GE Healthcare) which was made compatible with the Gateway system by insertion of the Gateway cassette Reading frame A (RfA) into the MCS. It allows for the IPTG-inducible expression of a N-terminally GST- tagged protein under control of a tac promoter and the lac operator (R. Lilischkis). GW-pGST-PARP10 (1- These vectors were created by a LR reaction with GW-pGST 255), (206-459), (408- and the corresponding pDONR/Zeo entry vectors (K. Montzka). 649), (600-868), (818- 1025), (818-1025) GW GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-995), (818-975), and the corresponding pDONR/Zeo entry vectors (H. (818-932), (818-891) Schuchlautz). GW-pGST-PARP10-C30 These vectors were created by a LR reaction with GW-pGST (996-1025), and the corresponding pDONR/Zeo entry vectors (H. -C50 (976-1025) Schuchlautz). GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025) I987E, and the corresponding pDONR/Zeo entry vectors (H. T999E, E1005A Schuchlautz). Experimental Procedures 106

GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025)GW E1005A, and the corresponding pDONR/Zeo entry vectors (H. D1013/1014/1024A, Schuchlautz). E1005A/D1013/ 1014/1024A, R1009/1021K GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025) P985A, and the corresponding pDONR/Zeo entry vectors (H. D980A/I982S, Schuchlautz). D980A/I982S/P985A, delC983, del981-984 GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025) E866A, and the corresponding pDONR/Zeo entry vectors (H. E870A,E877A,E882A Schuchlautz). GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025)GW and the corresponding pDONR/Zeo entry vectors (H. E866A,E870A, E877A, Schuchlautz). E882A GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025) E866Q, and the corresponding pDONR/Zeo entry vectors (H. E870Q,E877Q, E882Q Schuchlautz). GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025)GW and the corresponding pDONR/Zeo entry vectors (H. E866Q,E870Q, E877Q, Schuchlautz). E882Q GW-pGST-PARP10 These vectors were created by a LR reaction with GW-pGST (818-1025)GW and the corresponding pDONR/Zeo entry vectors (H. E866A/E870A, Schuchlautz). E870A/E877A, E877A/E882A, E866A/E870A/E877A, E866A/E870A/E882A, E870A/E877A/E882A GW-GST-mPARP14cat These vectors were created by a LR reaction with GW-pGST (1591-1817), and the corresponding pDONR/Zeo entry vectors (H. -PARP13cat (717-902), Schuchlautz). -PARP7cat (463-657), -PARP12cat (548-701) pGEX-2T-ARH1 This vector allows for the expression of a N-terminally GST- tagged ARH1 (Paul O. Hassa, EMBL Heidelberg). pGEX-2T-ARH2 This vector allows for the expression of a N-terminally GST- tagged ARH2 (Paul O. Hassa, EMBL Heidelberg). pGEX-2T-ARH3 This vector allows for the expression of a N-terminally GST- tagged ARH3 (Paul O. Hassa, EMBL Heidelberg). pGEX-2T-PARGcat This vector allows for the expression of the N-terminally GST- tagged catalytic domain (aa380-977) of bovine PARG (Paul O. Hassa, EMBL Heidelberg). pET24b-TEV-H The cDNA encoding the TEV protease was cloned into the MCS of pET24b (Novagen) (Brian H. Shilton, UWO London, Canada).

Experimental Procedures 107

Cloning vectors / Gateway entry vectors pDONR/Zeo This vector allows for the recombination of attB-PCR products into its attP1 and attP2 sites replacing the Gateway cassette containing the negative selective ccdB gene. M13 priming sites allow sequencing of the cloned PCR product in both directions. The EM7 promoter drives expression of the Zeocin resistance genes used for selection in E. coli. rrnB T1 and T2 transcription terminators protect the cloned gene from expression by vector- encoded promoters (Invitrogen). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (1-255) pDONR/Zeo and an attB-PCR product covering aa 1-255 of PARP10 (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (206-459) pDONR/Zeo and an attB-PCR product covering aa 206-459 of PARP10 (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (408-649) pDONR/Zeo and an attB-PCR product covering aa 408-649 of PARP10 (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (600-868) pDONR/Zeo and an attB-PCR product covering aa 600-868 of PARP10 (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-1025) pDONR/Zeo and an attB-PCR product covering aa 818-1025 of PARP10 (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-1025) pDONR/Zeo and an attB-PCR product covering aa 818-1025 of GW PARP10-G888W (K. Montzka). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (1-459) pDONR/Zeo and an attB-PCR product covering aa 1-459 of PARP10 (L. Milke). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (408-868) pDONR/Zeo and an attB-PCR product covering aa 408-868 of PARP10 (L. Milke). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (257-1025) pDONR/Zeo and an attB-PCR product covering aa 257-1025 of PARP10 (L. Milke). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (552-1025) pDONR/Zeo and an attB-PCR product covering aa 552-1025 of PARP10 (L. Milke). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10-C30 (996- pDONR/Zeo and an attB-PCR product covering aa 996-1025 of 1025) PARP10 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10-C50 (976- pDONR/Zeo and an attB-PCR product covering aa 976-1025 of 1025) PARP10 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-995) pDONR/Zeo and an attB-PCR product covering aa 818-995 of PARP10 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-975) pDONR/Zeo and an attB-PCR product covering aa 818-975 of PARP10 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-932) pDONR/Zeo and an attB-PCR product covering aa 818-932 of PARP10 (H. Schuchlautz). Experimental Procedures 108

pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP10 (818-891) pDONR/Zeo and an attB-PCR product covering aa 818-891 of PARP10 (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers I987E I987E_for/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers T999E P10T182E_for/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers E1005A PARP10E1005A_for/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW by site-directed mutagenesis using the primers GW E1005A PARP10E1005A_for/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW by site-directed mutagenesis using the primers GW D1013/1014/ D1013/D1014A_for and D1024A_for (H. Schuchlautz). 1024A pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW by site-directed mutagenesis using the primers GW R1009/1021K P10R192K_for and P10R204K_for (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E1005A by site-directed mutagenesis using the GW E1005A/ D1013/ primers P10R192K_for and P10R204K_for (H. Schuchlautz). 1014/1024A pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers P985A P985Afor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers C981S/C983S P10C981SC983Sfor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers D980A/I982S D980AI982Sfor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers D980A/I982S/P985A D980AI982SP985Afor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers delC983 del983neu_for/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers del981- del981-984 984_for/rev (H. Schuchlautz). pDONR/Zeo- These vectors were obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers E866A, E870A, E886Afor/rev, E870Afor/rev, E877Afor/rev, E882Afor/rev (H. E877A, E882A Schuchlautz). pDONR/Zeo- These vectors were obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW by site-directed mutagenesis using the primers GW E866A, E870A, E886Afor/rev, E870Afor/rev, E877Afor/rev, E882Afor/rev (H. E877A, E882A Schuchlautz). Experimental Procedures 109

pDONR/Zeo- These vectors were obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) by site-directed mutagenesis using the primers E866Q, E870Q, E886Qfor/rev, E870Qfor/rev, E877Qfor/rev, E882Qfor/rev (H. E877Q, E882Q Schuchlautz). pDONR/Zeo- These vectors were obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW by site-directed mutagenesis using the primers GW E866Q, E870Q, E886Qfor/rev, E870Qfor/rev, E877Qfor/rev, E882Qfor/rev (H. E877Q, E882Q Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E870A by site-directed mutagenesis using the GW E866A/E870A primers E866Afor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E870A by site-directed mutagenesis using the GW E870A/E877A primers E877Afor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E877A by site-directed mutagenesis using the GW E877A/E882A primers E882Afor/rev (H. Schuchlautz). pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E866A/E870A by site-directed mutagenesis using GW the primers E877Afor/rev (H. Schuchlautz). E866A/E870A/E877A pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E866A/E870A by site-directed mutagenesis using GW the primers E882Afor/rev (H. Schuchlautz). E866A/E870A/E882A pDONR/Zeo- This vector was obtained from pDONR/Zeo-PARP10 (818- PARP10 (818-1025) 1025) GW E870A/E877A by site-directed mutagenesis using GW the primers E882Afor/rev (H. Schuchlautz). E870A/E877A/E882A pDONR/Zeo- This entry vector was created by a Gateway BP reaction with mPARP14cat (1591- pDONR/Zeo and an attB-PCR product covering aa 1591-1817 1817) of murine PARP14 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP13cat (717- pDONR/Zeo and an attB-PCR product covering aa 707-902 of 902) human PARP13 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP7cat (463-657) pDONR/Zeo and an attB-PCR product covering aa 463-657 of human PARP7 (H. Schuchlautz). pDONR/Zeo- This entry vector was created by a Gateway BP reaction with PARP12cat (548- pDONR/Zeo and an attB-PCR product covering aa 548-701 of 701) human PARP12 (H. Schuchlautz).

4.4 Antibodies

-Actin (C4) Monoclonal mouse IgG1 antibody recognizing an epitope that has been conserved in actins from human skeletal muscle to plants (MP Biomedicals). -EEA1 (cl.14) Monoclonal mouse IgG1 antibody raised against rat EEA1 (BD Biosciences). -GM130 (cl. 35) Monoclonal mouse IgG1 antibody raised against the C-terminus of rat GM130 (BD Biosciences). -GFP (600-301- Monoclonal mouse IgG2a antibody raised against full length 215) GST-GFP (Rockland). Experimental Procedures 110

-HA (3F10) Monoclonal rat IgG1 antibody that recognizes the HA peptide sequence (YPYDVPDYA) derived from the influenza hemagglutinin protein, even when the HA peptide is introduced into unrelated recombinant proteins by “epitope tagging”. (Roche). -mouse IgG (H+L) Conjugated secondary antibody from goat recognizing the heavy Alexa 555- and light chains of murine IgG antibodies (Invitrogen). conjugated (A21425) -mouse IgG (H+L) Conjugated secondary antibody from goat recognizing the heavy Cy2-conjugated and light chains of murine IgG antibodies (Jackson Immuno (115-225-003) Research). -mouse IgG (H+L) Conjugated secondary antibody from donkey recognizing the Cy3-conjugated heavy and light chains of murine IgG antibodies (Jackson (715-165-151) Immuno Research). -mouse IgG + IgM Conjugated secondary antibody from goat recognizing the heavy (H+L) HRP- and light chains of murine IgG and IgM antibodies (Jackson conjugated (115- Immuno Research). 036-068) -PARP10 (5H11) Monoclonal rat antibody raised against GST-PARP10 (1-907) (Yu et.al., 2005). -PARP10 (891- Polyclonal serum from rabbits immunized against GST-PARP10 6/890-6) (1-907) (Yu et.al., 2005). 891-0/890-0 Control bleeds corresponding to 891-6/890-6. -PARP1 (1835238) Polyclonal serum from rabbits immunized against full length recombinant PARP1 (Roche). -PAR (10H) Monoclonal mouse IgG3 antibody raised against purified poly- ADP-ribose (Calbiochem). -PAR (96-10-04) Polyclonal rabbit antibody raised against poly-ADP-ribose with methylated BSA. It is described to recognize PAR of 6-100 bases in size (Alexis Biochemicals). -phospho-Rb Polyclonal rabbit antibodies raised against a synthetic KLH- (Ser780, coupled phospopeptide corresponding to residues around Ser807/811) (9307, Ser780 and Ser807/811 of human Rb (Cell Signaling). 9308)

-Rab5 (cl. 1) Monoclonal mouse IgG2 antibody raised against human Rab5 (BD Biosciences). -rabbit IgG + IgM Conjugated secondary antibody from goat recognizing the heavy (H+L) HRP- and light chains of rabbit IgG and IgM antibodies (Jackson conjugated (111- Immuno Research). 035-144) -rat IgG+IgM (H+L) Conjugated secondary antibody from goat recognizing the heavy Cy3-conjugated and light chains of rat IgG and IgM antibodies (Jackson Immuno (112-166-068) Research). -rat IgG+IgM (H+L) Conjugated secondary antibody from goat recognizing the heavy FITC-conjugated and light chains of rat IgG and IgM antibodies (Jackson Immuno (112-096-068) Research). -rat IgG + IgM Conjugated secondary antibody from goat recognizing the heavy (H+L) HRP- and light chains of rat IgG and IgM antibodies (Jackson Immuno conjugated (112- Research). 035-068) -Rb (C-15) Polyclonal rabbit antibody with an epitope mapping at the C- terminus of human Rb (Santa Cruz).

Experimental Procedures 111

4.5 Work with nucleic acids 4.5.1 DNA preparation DNA was extracted from transformed bacteria following the manufacturer’s instructions using the Qiagen Plasmid Maxi Kit (Qiagen) for preparative purposes and the Zippy Plasmid Miniprep Kit (Zymo Research) for small-scale preparations.

4.5.2 RNA preparation Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.

4.5.3 Enzymatic manipulation of plasmid DNA 10x Magic Buffer: 200 mM Tris pH 7.5 700 mM NaCl 200 mM KCl 100 mM MgCl2 0.5 mM spermine 0.125 mM spermidine

Calf Intestine Alkaline Phosphatase (Fermentas)

T4 DNA Ligase (Fermentas)

Plasmid DNA was digested in 20 l reaction volume using restriction enzymes (Fermentas) with the appropriate buffers for 1h to o/n at the appropriate temperature, usually 37°C. Double digests were routinely performed in magic buffer. De- phosphorylation of terminal 5’-phosphates was achieved by incubation with calf intestine alkaline phosphatase (Fermentas) for 1h. DNA was ligated using T4 DNA ligase (Fermentas). Sticky-end ligations were carried out for 1-2h at ambient temperature. For blunt-end ligations, 5% PEG2000 was included in the reaction, and the reaction was incubated o/n at 16°C.

4.5.4 Agarose gel electrophoresis TBE: 89 mM Tris-Base 89 mM boric acid 2 mM EDTA

10x DNA Loading Buffer: 50 mM Tris pH 8.0 50 mM EDTA 50% (v/v) glycerol 0.25% (w/v) bromophenol blue 0.25% (w/v) xylene cyanol

Agarose Low EEO (Sigma) NuSieve GTG Agarose (Biozym Scientific)

Gel electrophoresis of DNA was performed as described (Sambrook et al., 1989) in 0.8-2% agarose gels using low EEO agarose (Sigma) or in 3.5-4% agarose gels using NuSieve GTG Agarose (Biozym Scientific ) for short DNA fragments and hybridized oligonucleotides.

Experimental Procedures 112

4.5.5 Gel extraction of DNA DNA was recovered from agarose gels after electrophoresis using the Zippy Gel DNA Recovery Kit (Zymo Research) according to the manufacturer’s instructions.

4.5.6 Gateway cloning The Gateway technology (Invitrogen) is a universal cloning method based on the site- specific recombination properties of bacteriophage lambda. It allows for the rapid and efficient movement of DNA sequences into multiple vector systems. First, the desired target DNA is amplified by PCR using primers possessing the recombination sites (att sites). Second, the PCR product flanked by att sites is recombined into a so-called entry vector. Finally, this entry vector can be used to recombine into the destination vector of interest depending on the intended use. Gateway cloning was performed according to the manufacturer’s instructions with slight modifications. For PCR amplification, either an equal mixture of recombinant Taq polymerase (Fermentas) and cloned Pfu polymerase (Fermentas) or 1-2 U of Phusion polymerase (Finnzymes) was used. The recombination reactions were carried out o/n with half the amount of reagents as recommended.

4.5.7 Site-directed mutagenesis Primers for site-directed mutagenesis were designed using the online available QuikChange Primer Design Program (Stratagene). The mutagenesis of single or adjacent base pairs was carried out using the QuikChange Site-directed Mutagenesis Kit (Stratagene) following the manufacturer’s instructions. For the introduction of several mutations, the QuikChange Multi Site-directed Mutagenesis Kit (Stratagene) was used. All obtained plasmids were checked for correct amplification by sequencing of the whole insert DNA.

4.5.8 Generation of pSuper-based siRNA constructs siRNA oligonucleotides targeting the desired sequence were designed using the online available program siDirect (genomics.jp/sidirect). After using the BLAST algorithm sequences being specific for both strands were selected. An oligonucleotide was designed according to the following scheme: 5’ GAT CCC C target sequence TTC AAG AGA complementary sequence TTT TTG GAA A 3’. Another oligonucleotide was designed according to the following scheme: 5’ AGC TTT TCC AAA AA target sequence TCT CTT GAA complementary sequence GGG 3’. The hybridized oligos were cloned into pSuper (Brummelkamp, 2002) digested with BglII and HindIII.

4.6 Work with prokaryotic cells 4.6.1 Bacteria strains E.coli XL10-Gold Tetr D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi- (Stratagene) 1 recA1 gyrA96 relA1 lac Hte [F’ proAB lacIqZDM15 Tn10 (Tetr) Amy Camr] - - E.coli DH5 F 80dlacZM15(lacZYA-argF) U169 recA1 endA1 hsdR17(rk + - (Invitrogen) mk ) phoA supE44 thi-1 gyrA96 relA1 - - - E.coli DB3.1 F gyrA462 endA (sr1-recA) mcrB mrr hsdS20 (rB mB ) supE44 (Invitrogen) ara14 galK2 lacY1 proA2 rpsL20 (StrR) xyl5 - leu mtl1 - – – r E.coli BL21(DE3) B F dcm ompT hsdS(rB mB ) gal (DE3) [pLysS Cam ] pLysS (Stratagene)

Experimental Procedures 113

4.6.2 Materials for work with prokaryotic cells LB Medium: 1% (w/v) tryptone 0.5% (w/v) yeast extract 1% (w/v) NaCl pH 7.0

Low Salt LB Medium: 1% (w/v) tryptone 0.5% (w/v) yeast extract 0.5% (w/v) NaCl pH 7.5

TB Medium: 1.2% (w/v) Tryptone 2.4% (w/v) yeast extract 4% (v/v) glycerol 17 mM KH2PO4 74 mM K2HPO4

Agar plates (Amp/Kan): LB Medium 1.5% (w/v) Bacto Agar (Difco) 100 g/ml ampicillin or 30 g/ml kanamycin

Agar Plates (Zeo): Low Salt LB Medium 1.5% (w/v) Bacto Agar (Difco) 50 g/ml zeocin (Invitrogen)

4.6.3 Protocols for work with prokaryotic cells 4.6.3.1 Bacterial Transformation After thawing 100 μl chemically competent bacteria were transferred to a 14 ml round- bottom tube (Falcon) and mixed with plasmid DNA, usually 100-500 ng. After up to 30 minutes on ice bacteria were heat-shocked for 45 seconds at 42°C and immediately placed back on ice. Subsequently, 1 ml of LB medium was added and bacteria incubated for 1h at 37°C. Bacteria were pelleted by centrifugation, re-suspended in 100 l of LB medium and plated on LB agar plates containing the appropriate antibiotic. Plates were incubated o/n at 37°C until bacterial colonies got visible.

4.6.3.2 Purification of GST fusion proteins TNE Buffer: 20 mM Tris pH 8.0 150 mM NaCl 1 mM EDTA 5 mM DTT 1 mM Pefabloc SC (Roche) 14 μg /ml aprotinin

GST Wash Buffer: 100 mM Tris pH 8.0 120 mM NaCl

GST Elution Buffer: 100 mM Tris pH 8.0 120 mM NaCl 20 mM glutathione

Experimental Procedures 114

Two to three colonies of transformed bacteria were used to inoculate a starter culture of usually 50 ml LB medium supplemented with 0.4-1.0% (w/v) Glucose and the appropriate antibiotic which was grown o/n at 37°C. The main culture was started by dilution of the starter culture at least 1:30 in the same type of medium. The culture was grown at 37°C until an OD600 0,5-0,7 was reached. Afterwards expression of the GST fusion protein was induced by addition of 0.5-1.0 mM IPTG. Expression was allowed for 2-3h at 37°C or for o/n at ambient temperature. Subsequently, bacteria were pelleted by centrifugation at 3,500 xg, re-suspended in ice-cold TNE buffer and lysed for 30 min on ice in the presence of 100 μg/ml lysozyme. Cells were solubilized by sonication and lysates cleared from cell debris by centrifugation at 10,000 xg for 30 min. The supernatant was incubated with 500 l equilibrated Glutathione Sepharose 4B beads (Amersham Biosciences) per 30 ml of lysate for 1h at 4°C. Beads were washed three times with PBS and transferred to a 0.8x4 cm chromatography column (Bio-Rad). After washing with 1 ml of GST wash buffer bound proteins were eluted in three fractions with 300 l of GST elution buffer per fraction. Aliquots of the eluted proteins were subjected to SDS-PAGE and subsequent Coomassie staining in order to estimate the concentration of the purified proteins in comparison to a BSA standard series.

4.6.3.3 Purification of His-tagged TEV protease Buffer A: 20 mM HEPES pH 7.5 300 mM NaCl 10% (v/v) glycerol 0.1% (v/v) NP-40 5 mM imidazole 14 μg/ml aprotinin

Buffer B: 20 mM HEPES pH 7.5 200 mM NaCl 10% (v/v) glycerol 0.1% (v/v) NP-40 10 mM EDTA 10 mM DTT 14 μg/ml aprotinin

Buffer C: 50 mM Tris pH 8.0 1 M NaCl 10% (v/v) glycerol 0.5 mM DTT 1 mM EDTA

E. coli BL21 (DE3) transformed with vector pET24b-TEV-H were used to inoculate a culture in TB medium. Expression was induced by addition of 0.4-1.0 mM IPTG. After incubation for 3-4h at 37°C cells were harvested and re-suspended in buffer A. After incubation with lysozyme the lysate was sonicated well and cleared by centrifugation. His-tagged TEV protease was pulled out with TALON™ Metal Affinity Resin (BD Biosciences), washed extensively with buffer A and eluted with buffer B. Eluted TEV protease was dialysed o/n against the high salt buffer C, aliquoted and stored at -80°C. All purification steps from lysis on were carried out on ice or at 4°C to prevent denaturation of the thermally instable TEV protease. The amount of TEV protease needed to provide efficient cleavage was determined empirically for every single charge.

Experimental Procedures 115

4.7 Work with eukaryotic cells (cell culture) 4.7.1 Eukaryotic cell lines HEK 293 This is an adherent epithelial cell line derived from human (ATCC CRL-1573) embryonic kidney (HEK) cells transformed with Adenovirus 5 DNA. The Ad5 insert is integrated into chrosome 19q13.2. It is a hypotriploid cell line with a modal chromosome number of 64, occurring in 30% of cells. HeLa This is an adherent epithelial cell line derived from a cervical (ATCC CCL-2) adenocarcinoma of a 31 year old black female. 100% aneuploidy is observed with a modal chromosome number of 82. HeLa cells have been reported to contain HPV-18 sequences. P53 expression was reported to be low, and normal levels of pRB were found. U-937 This monocytic suspension cell line was derived from (ATCC CRL-1593.2) malignant cells obtained from the pleural effusion of a 37 year old male patient with histiocytic lymphoma. U-937 cells can be induced to terminal monocytic differentiation by phorbol esters, vitamin D3, -interferon, TNF, and retinoic acid. COS-7 This is an African green monkey (Cercopithecus aethiops) (ATCC CRL-1651) kidney fibroblast-like cell line suitable for transfection by vectors requiring expression of SV40 T antigen. This line contains T antigen, retains complete permissiveness for lytic growth of SV40, supports the replication of ts A209 virus at 40C, and supports the replication of pure populations of SV40 mutants with deletions in the early region. The line was derived from the CV-1 cell line (ATCC CCL-70) by transformation with an origin defective mutant of SV40 which codes for wild type T antigen. U-2 OS This is an adherent epithelial cell line derived from a (ATCC HTB-96) moderately differentiated osteosarcoma of a 15 year old girl. It is chromosomally highly altered, with chromosome counts in the hypertriploid range. Flp-In T-REx-293 This cell line was derived from HEK293. It expresses the Tet (Invitrogen #R780-07) repressor from pcDNA6/TR and contains a single integrated Flp Recombination Target (FRT) site from pFRT/lacZeo resulting in expression of a lacZ-Zeocin fusion gene. This cell line can be used to generate a tetracycline-inducible expression cell line by cotransfecting the pcDNA5/FRT/TO expression vector containing your gene of interest and the Flp recombinase expression plasmid, pOG44. Flp-In T-REx-293- This stably transfected cell lines were generated by pcDNA5/FRT/ transfection of Flp-In T-REx-293 cells with pOG44 and TO/C-TAP, pcDNA5/FRT/TO/C-TAP, C-TAP-PARP10, C-TAP-PARP10- C-TAP-PARP10, G888W, N-TAP-PARP10. Stable cell lines were obtained by C-TAP-PARP10- selection with hygromycin B and Blasticidin S. G888W, N-TAP- PARP10

Experimental Procedures 116

4.7.2 Materials for cell culture work DMEM (Gibco) with 4,5 g/l Glucose PBS: 140 mM NaCl 2.6 mM KCl 2 mM Na2HPO4 1.45 mM KH2PO4

Blasticidin S (Invivogen) 10 mg/ml Doxycyclin (Sigma) 1 mg/ml Hygromycin B (Roche) 50 mg/ml Penicillin / Streptomycin (Seromed) 10.000 Units/ml / 10.000 g/ml Trypsin / EDTA (Seromed) 0.5 / 0.02% (w/v) in PBS FCS (Gibco) Tissue culture dishes (Sarstedt) 6 cm, 10 cm, 15 cm Tissue culture plates (TPP) 6-Well 1 ml cyrotubes (Nalgene)

Culture conditions:

All cell lines were cultured under humidified atmosphere of 5% CO2 at 37°C. HEK 293, HeLa, and COS-7 cells were cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) Penicillin/Streptomycin. U-937 cells were cultured in RPMI-1640 supplemented with 10% (v/v) FCS and 1% (v/v) Penicillin/Streptomycin. U-2 OS cells were cultured in RPMI-1640 supplemented with 10% (v/v) FCS and 1% (v/v) Penicillin/Streptomycin. Stably transfected Flp-In T-REx-293 cell lines were maintained in regular DMEM supplemented with 15 μg/ml Blasticidin S and 50 μg/ml hygromycin B.

4.7.3 Protocols for work with eukaryotic cells 4.7.3.1 Cryo-conservation and thawing of cells For cryo-conservation, cells grown to log phase were usually re-suspended in cryotubes in 1 ml FCS with 10% (v/v) DMSO at a density of 5-8x106. After incubation on ice for 30 min, the cryo tubes were wrapped with several layers of tissue paper, put into an insulated box and slowly cooled down for 16-48h at -80°C. Afterwards, the cryotubes were transferred to -150°C for long-term storage. For thawing of frozen cells, cryotubes were thawed in a water bath at 37°C. Immediately after melting of the ice crystals, cells were suspended in 10 ml fresh medium, pelleted by centrifugation, re-suspended in 10 ml fresh medium and transferred to an appropriate culture dish or flask.

Experimental Procedures 117

4.7.3.2 Transient transfection

A) calcium phosphate method

2x Hebs buffer: 274 mM NaCl 42 mM HEPES 9.6 mM KCl 1.5 mM Na2HPO4 pH 7.1

HEPES buffer: 142 mM NaCl 10 mM HEPES 6.7 mM KCl pH 7.3

250 mM CaCl2

The transient transfection of adherent cells was performed according to the calcium phosphate method (Bousset et al., 1994; Chen and Okayama, 1988). This method is based on the precipitate formation of calcium-phosphate-DNA complexes which are taken up by the cells through endocytosis. The calcium phosphate method was used to transfect HEK 293 cells. The day prior to the transfection, about 1x106 /4x105 cells were seeded on 10 cm/6 cm tissue culture dishes. For transfection, 20 μg/4 μg of total DNA was diluted in 500 l/200 μl 250 mM CaCl2 and incubated for up to 30 min at ambient temperature. Subsequently, 500 μl/200 μl 2x Hebs buffer was added in drops, the solution mixed by agitation and afterwards pipetted onto the cell monolayer. The dishes were swayed and put back into the incubator. After up to 6h incubation time, the cells were freed from residual precipitate by washing with 10 ml/4 ml HEPES buffer. Subsequently, fresh medium was added.

B) ExGen 500 transfection reagent (Fermentas)

ExGen 500 is a 22 kDa linear polyethylenimine that interacts with DNA to form small, highly diffusible complexes which are readily endocytosed. ExGen 500 was used for the transfection of HeLa cells according to the manufacturer’s instructions with an ExGen 500/DNA ratio of 3.3.

C) FuGENE 6 transfection reagent (Roche)

FuGENE 6 is a lipid-based transfection reagent. Lipids and DNA form complexes due to ionic interactions and are taken up by the transfected cells. FuGENE 6 was used for the transfection of COS-7 cells according to the manufacturer’s instructions with an FuGENE 6/DNA ratio of 3.

Experimental Procedures 118

4.7.3.3 Preparation of cell lysates Frackelton Buffer (F Buffer): 10 mM HEPES pH 7.5 50 mM NaCl 30 mM Na4P2O7 50 mM NaF 5 M ZnCl2 pH 7.05 0.2 % (v/v) Triton X-100 10% (v/v) glycerol 100 M Na3VO4 500 M Pefa Bloc (Roche) 0.025 U/ml 2-macroglobulin (Roche) 2.5 g/ml pepstatin A 2.5 g/ml leupeptin 150 M benzamidin 14 g/ml aprotinin 10 mM sodium ß-glycerophosphate

Cells were lysed in F buffer or TAP lysis buffer (see below) at a concentration of about 5x107 cells per ml of buffer. Adherent cells were incubated on ice for 5 min, scraped off, transferred into an Eppendorf tube and incubated on ice for additional 10 min. Suspension cells were incubated in an Eppendorf tube on ice for 20 min. Cell debris was spun down at 16,000 xg for 20 min at 4°C. The supernatant was transferred to a new Eppendorf tube and either used immediately for further analysis or stored at - 20°C.

4.7.3.4 Colony formation assay Methylene Blue Solution: 0.2 % (w/v) methylene blue in MeOH Puromycin 1 mg/ml

The day before transfection, 2.5 x105 HeLa cells were seeded onto 6 cm culture dishes. The cells were transfected using ExGen 500 with 5 g total plasmid DNA including 0.5 g pBabePuro for selection. 6 h after transfection the medium was replaced. After additional 3 h puromycin was added to a final concentration of 2 g/ml. Cells were selected for 16 h and supplied with fresh medium. Cells were let grown for additional 3-6 days until the cells transfected with a control plasmid reached a confluency of about 90%. Cells were washed once with PBS and the formed colonies were stained with methylene blue solution for up to 30 min. After two washes with ddH2O the plates were air-dried and the colony formation documented with a scanner.

4.8 Work with proteins 4.8.1 Denaturing Discontinuous Polyacrylamide Gel Electrophoresis (SDS-PAGE) 2x/4x Sample Buffer: 160 mM / 320 mM Tris pH 6.8 20% / 40% (v/v) glycerol 10% / 8% (w/v) SDS 0.25% / 0.5% (w/v) BPB 100 mM / 200 mM ß-ME

Experimental Procedures 119

Running Buffer: 25 mM Tris base 250 mM glycine 0.1% (w/v) SDS

Protein Ladder: PageRuler Prestained Protein Ladder, 11-170 kDa (Fermentas) PageRuler Protein Ladder, 10-200 kDa (Fermentas)

The denaturing SDS-PAGE allows for the separation of complex protein mixtures according to the molecular size of the proteins. SDS binds quantitatively to the denatured proteins and generates a negative net charge of the proteins due to its anonic properties. Therefore, proteins can be separated solely by their molecular weight during their electrophoretically enforced movement towards the anode through a polyacrylamide gel matrix. The discontinuous SDS-PAGE was essentially performed according to Laemmli (Laemmli, 1970) with a 5% stacking gel and a 10-20% separating gel.

4.8.2 Western Blot Semi-dry Transfer Buffer: 25 mM Tris base 192 mM glycine 20% (v/v) methanol

Tank Blot Transfer Buffer: 25 mM Tris base 192 mM glycine 0.1% (w/v) SDS

0.2% (w/v) Ponceau S in 3% (v/v) TCA

Proteins separated by SDS-PAGE can afterwards be transferred electrophoretically onto nitrocellulose or PVDF membranes. During the transfer the SDS is mainly removed from the proteins resulting in at least partially renaturation of the proteins which allows for subsequent immunodetection (see below). The semi-dry transfer includes a “sandwich” of gel and membrane surrounded by thick Whatman papers soaked in semi-dry transfer buffer. This sandwich is placed in a blotting chamber between two electrode plates. The Western Blot was essentially performed according to Towbin (Towbin et al., 1979). For semi-dry transfer, proteins were transferred from the gel to the membrane for 1h at 2 mA per cm2 of membrane size. In order to fix the proteins on the membrane and to control transfer efficiency, the membrane was routinely stained with Ponceau S for 1-2 min and afterwards washed twice with dH2O.

4.8.3 Immunodetection of proteins PBS-T: PBS 0.05% (v/v) Tween-20

Blocking Buffer: PBS-T 5% (w/v) skim milk powder

Experimental Procedures 120

ECL solutions (Pierce)

After blotting proteins onto a membrane, they can be detected by specific antibodies. This immunoblot procedure involves the blocking of unspecific binding sites on the membrane using skim milk or BSA, the specific binding of a primary antibody targeted against the protein of interest, the specific binding of a secondary antibody targeted against the invariable part of the primary antibody which is coupled to horseradish peroxidase (HRP), and finally the addition of a chemoluminescent substrate which is turned over by the HRP under emission of light. This emission can be detected by a highly sensitive camera system or by X-ray films. Routinely, blocking was achieved by incubation of the membrane in blocking buffer for at least 30 min. The primary antibody was incubated for 2h at ambient temperature or o/n at 4°C. After at least five washes in PBS-T with minimal 5 min per wash step, the secondary antibody was incubated for 45-60 min at ambient temperature. Prior to detection, at least three rounds of washing in PBS-T were performed. The detection was carried out with a computer-assisted camera (LAS-3000, Fuji).

4.8.4 Rapid Coomassie Staining Fixing Solution: 25% (v/v) isopropanol 10% (v/v) acetic acid

Staining Solution: 10% (v/v) acetic acid 0.006% (w/v) CBB G-250 (Bio-Rad)

Destaining Solution: 10% acetic acid

Coomassie stain stains proteins unspecifically through interaction with cationic and hydrophobic amino acid residues. After SDS-PAGE, the gel was fixed for 10-15 min and afterwards stained for 2-3h under slight agitation. In order to reduce background staining, the gel was destained either for 1-2h in destaining solution or o/n in dH2O.

4.8.5 Mass-spectrometric analysis 0.5 M Iodoacetamide Gel-Code Blue Stain Reagent (Pierce)

In order to avoid possible keratin contaminations, all solutions were prepared with Milli- Q water and sterilized by filtration through a 0.22 m filter (Millipore). As far as possible, all working steps were carried out under clean bench conditions. For preparing proteins samples for mass-spectrometric analysis, the samples were diluted in 2x sample buffer, boiled for 5 min at 95°C and cooled down to 55°C. After addition of iodoacetamide to a final concentration of 100 mM, the samples were incubated az 55°C for 10 min. Subsequently, the samples were subjected to SDS- PAGE. After electrophoresis, the gel was stained with Gel-Code Blue following the manufacturer’s instructions. For further analysis, the gel was shrink-wrapped and shipped to David W. Litchfield (University of Ontario, London, Canada).

Experimental Procedures 121

4.8.6 Tandem Affinity Purification TAP Lysis Buffer: 50 mM Tris pH 7.5 150 mM NaCl 1 mM EDTA 10% (v/v) glycerol 1% (v/v) NP-40 1 mM DTT 100 μM sodium vanadate 14 g/ml aprotinin 4 μM leupeptin 0.5 mM PMSF

TEV Buffer: 50 mM Tris pH 7.5 150 mM NaCl 0.5 mM EDTA 1 mM DTT

Calmodulin Binding Buffer: 10 mM Tris pH 7.5 150 mM NaCl 0.2% (v/v) NP-40 1 mM magnesium acetate 2 mM calcium chloride 1 mM imidazole 10 mM -ME

Calmodulin Wash Buffer : 50 mM ammonium bicarbonate pH 8.0 75 mM NaCl 1 mM magnesium acetate 1 mM imidazole 2 mM calcium chloride

Calmodulin Elution Buffer : 50 mM ammonium bicarbonate pH8.0 25 mM EGTA

IgG affinity matrix: IgG Sepharose 6 Fast Flow (Amersham Biosciences)

Calmodulin affinity matrix: Calmodulin Sepharose 4B (Amersham Biosciences)

Tandem affinity purification (TAP) allows for the highly selective purification of protein complexes under native conditions. This is reached by two successive purification steps: the first one involves the binding of Protein A to IgG Sepharose beads from which the purified proteins are eluted through cleavage of the Protein A-tag by TEV protease. The second step is the binding of the residual Calmodulin binding peptide to Calmodulin Sepharose beads. Finally, purified proteins are eluted using EGTA which disrupts the Ca2+-dependent binding to the Calmodulin column. Expression of TAP fusion proteins from stable cell lines was induced by 1 g/ml doxycycline for 10-14h. Cells were harvested by centrifugation at 200 xg, washed once with ice-cold PBS and pelleted again. All following steps were carried out at 4°C. Cells were re-suspended in 15 ml lysis buffer per 500 ml of suspension culture and lysed for Experimental Procedures 122

30 minutes under slight agitation. Lysate was cleared by centrifugation at 15,000 xg for 20 minutes. The supernatant was incubated with 125 l equilibrated IgG Sepharose 6 FF (Amersham Biosciences) per 15 ml lysate for 1h under rotation. Beads were pelleted at 200 xg for 2 minutes and washed three times with TEV buffer. Beads were re-suspended in five volumes of TEV buffer and approximately 200-500 ng of His- tagged TEV protease per 10 l beads were added. Cleavage was performed for 2h under permanent agitation. After TEV cleavage, the beads were pelleted and the supernatant was transferred to a new tube containing equilibrated Calmodulin Sepharose 4B (Amersham Biosciences) (equal volume as for IgG sepharose). IgG Sepharose was washed once with three volumes of calmodulin binding buffer, pelleted and the supernatant combined with the supernatant of the TEV cleavage. After addition of 1/200 volume of 1 M CaCl2, the pooled supernatants were incubated with the Calmodulin Sepharose for 90 minutes. Subsequently the beads were pelleted and washed three times with calmodulin wash buffer. Beads were re-suspended in two volumes of elution buffer and incubated for 20 minutes under permanent agitation. Beads were pelleted and the supernatant containing the purified proteins was transferred to a new tube avoiding transfer of beads and stored at -80°C. To avoid negative effects of EGTA on enzymatic activity in subsequent assays 25 mM MgCl2 was added prior to storage.

4.9 Enzymatic assays 4.9.1 PARP assay PARP Reaction Buffer: 50 mM Tris pH 8.0 0.2 mM DTT 4 mM MgCl2

[32P]-NAD+ 370 MBq/ml (10 Ci/l) (Amersham Biosciences) ß-NAD+ (Sigma)

A PARP assay detects the incorporation of radioactively labeled [32P]-NAD+ into substrate proteins mediated by enzymes of the ADP-ribosyltransferase class. PARP assays were carried out routinely in 30 l reaction volume at 30°C for 30 minutes in PARP reaction buffer using 50 M ß-NAD+ (Sigma) and 1 Ci 32P-NAD+ (Amersham Biosciences) if not indicated otherwise. 500 ng of enzyme and 1 g of substrates were used routinely. The reactions were stopped by addition of SDS sample buffer. For quantification of incorporated label either radioactive bands were cut out and activity measured by Cerenkov counting or reactions were stopped by addition of 500 l BSA (100 g/ml) and 530 l ice-cold TCA, precipitated material was spotted onto GF/C filter (Whatman), washed several times with 5% TCA and subsequently with 95% EtOH, air-dried and measured by Cerenkov counting. PARP1 used as positive control was baculo-derived. This protein was already associated with DNA resulting in full enzymatic activity and addition of DNA did not result in a further activation (our unpublished observation).

4.9.2 Analysis of ADP-ribosylation reaction products Diethyl ether Phenol/Chloroform/IAA (49:49:2) Alkaline TE Buffer: 10 mM Tris 1 mM EDTA pH 12.0

Experimental Procedures 123

Urea Loading Buffer: 50% (w/v) urea 25 mM NaCl 4 mM EDTA 0.02% xylene cyanol 0.02% bromophenol blue

Phosphodiesterase Buffer: 100 mM Tris pH 8.0 100 mM NaCl 15 mM MgCl2 1 mM sodium vanadate 100 nM okadaic acid

Phosphodiesterase I (USB) 1 mg/ml in 100 mM Tris pH 8.0, 100 mM NaCl, 15 mM MgCl2 and 50% glycerol

TLC plates PEI-F cellulose 20 cm x20 cm (Merck)

Solvents A: isobutyric acid / 25% NH4OH / H20 (50/1.1/28.9, by volume)

Solvents B: 0.1 M sodium phosphate pH 6.8 / ammonium sulfate / n-propanol (100/60/2, v/w/v)

PARP assays were carried out as decribed above using 5 Ci 32P-NAD+ and indicated amounts of ß-NAD+. Reactions were stopped by addition of 500 l BSA (100 g/ml) and 530 l ice-cold TCA. Analysis of polymer length by PAGE was carried out as described previously (Panzeter and Althaus, 1990). Briefly, TCA-precipitated proteins were washed twice with diethyl ether and re-suspended in alkaline TE buffer. ADP-ribose modifications of acidic amino acid residues were detached from the proteins during a 3h incubation at 60°C. Subsequently, samples were extracted with Phenol/Chloroform/IAA, and the aqueous phase dried in a Speed-Vac. Samples were dissolved in urea loading buffer and, after pre-electrophoresis for 1h at 55 W (constant power), loaded onto a 20% polyacrylamide gel (60 cm x20 cm x0.15 mm). Electrophoresis at 55 W was carried out until the bromophenol blue dye had migrated about three-fourths of the entire gel length. The gel was transferred to a thin Whatman paper and exposed to X-ray films at -80°C. Analysis of the ADP-ribosylation reaction products by 2D-TLC after hydrolysis with phosphodiesterase I (purchased from USB) was carried out as reported previously (Keith et al., 1990). Briefly, samples were treated as described above for analysis of polymer length. But after drying the samples in a Speed-Vac, they were dissolved in 30 l of phosphodiesterase buffer. After addition of 1 l of phosphodiesterase I, ADP- ribosylation reaction products were hydrolyzed for 3.5h at 37°C. In order to separate the hydrolysis products, 3 l of the reaction were spotted onto a TCL plates. The development was performed in two dimension using solvents A in the first dimension and solvents B in the second dimension. Dried TLC plates were exposed to X-ray films at -80°C.

4.9.3 PARG/ARH assay PARG/ARH Buffer: 50 mM potassium phosphate buffer pH 7.5 10 mM MgCl2 5 mM DTT Experimental Procedures 124

Proteins were immobilized on beads, either glutathione agarose or Protein A beads coupled with the appropriate antibody, and auto-modified in a standard PARP assay with 32P-NAD+ as described above. The auto-modified proteins were pelleted by centrifugation, the beads washed two times with PARG/ARH buffer and re-suspended in 40 l PARG/ARH buffer. After addition of 1 g GST or GST-PARGcat, -ARH1, - ARH2 or -ARH3, the reaction was incubated for 2h at 37°C to allow for the de-ADP- ribosylation of the auto-modified proteins. The reaction was stopped by addition of 15 l 4x sample buffer and subjected to SDS-PAGE. After drying the gel, residual radioactivity was determined via exposure to X-ray films at -80°C.

4.9.4 Phosphatase assay Phosphatase Buffer: 50 mM Tris Ph 8.0 1 mM MgCl2 100 M sodium vanadate 50 mM NaF 10 mM sodium ß-glycerophosphate 50 nM okadaic acid

Shrimp Alkaline Phosphatase 5U/l (Roche)

Potentially phosphorylated proteins were immunoprecipitated using Protein A or Protein G beads and the appropriate antibodies. The beads were washed two times with the used lysis buffer and two times with phosphatase buffer. Subsequently, the beads were re-suspended in 30 l phosphatase buffer and 5U of phosphatase were added or not added in a mock-treated sample, respectively. The reaction was incubated for 30 min at 30°C. Afterwards, the reaction was stopped by addition of 15 l 4x sample buffer and the mixture subjected to SDS-PAGE. The phosphorylation state of the proteins was checked by phospho-specific antibodies, if possible.

4.10 Microscopy techniques 4.10.1 Immunocytochemistry Glass coverslips PBS Fixing Solution: 3.7% PFA in PBS Permeabilizing Solution: 0.1% Triton-X100 in PBS Blocking Solution: 1% BSA in Permeabilizing Solution Antibody Solution: 0.2% BSA in PBS Hoechst 33258 10 mg/ml Mowiol 4-88

Cells grown on glass coverslips were washed twice with PBS and fixed for 15-30 min at ambient temperature depending on the cell line. The cells were washed with PBS and afterwards with permeabilizing solution. Blocking of unspecific binding sites was achieved by 30 min incubation with blocking solution at ambient temperature. The primary antibody directed against the protein to be visualized was diluted in antibody solution and incubated on the cells for 45-60 min in a humid dark box at 37°C. Unbound antibody was washed away by several rounds of washing with antibody solution. The secondary antibody conjugated to the desired fluorophore was diluted in antibody solution and incubated in the same way as the primary one. After washing with antibody solution, PBS and ddH20, Hoechst stain was diluted 1/5000 in ddH20 and Experimental Procedures 125

incubated for 1-2 min. After washing twice with ddH20, the coverslip was embedded in Mowiol 4-88. The next day stained cells were inspected by epifluorescence microscopy on an Olympus IX50 or by confocal microscopy on a Zeiss Axiovert LSM510.

4.10.2 Time-lapse microscopy For live-cell imaging, cells were seeded onto glass coverslips or glass-bottom dishes (Willco Wells) and transfected using FuGene reagent (Roche). Time-lapse microscopy was performed on a confocal Zeiss Axiovert LSM510 or on an epifluorescent microscope Zeiss Axiovert 200 (in collboration with A. Sechi) both equipped with an incubation chamber set to 37°C and a CO2 gas supply providing constantly 5% CO2.

4.10.3 iFLAP (inverted fluorescence localization after photobleaching) For iFLAP analysis, cells were seeded onto glass coverslips and transfected with plasmids expressing CFP/YFP fusion proteins using FuGene reagent (Roche). iFLAP was performed on a confocal Zeiss Axiovert LSM510 with a water-corrected C- Apochromat 63x objective (NA 1.2). The pinhole size was set to 200 m. YFP was excited with a 514 nm laser and detected with a BP 530-600 nm filter set. CFP was excited with a 458 nm laser and detected with a BP 480/20 nm filter set. For the measurement of iFLAP, the YFP fluorescence was repetitively bleached about every six seconds at maximum 514 nm laser intensity with 20 pulses within a defined ROI in the cytoplasm. After each bleaching, an image of the size 512x512 pixels was taken in the CFP and the YFP channel. A total of 120 frames were taken within about 12 minutes of measurement. An unbleached control cell situated next to the bleached cell was analyzed in parallel to control the potential unspecific effects caused by photobleaching. The pictures were filtered using the “median” and “lowpass 7x7” function and processed by the ratio function: Channel 1 (YFP) / Channel 2 (CFP) x (-4096) + 4096. The processed images were pseudo-colored with a rainbow palette. While blue color corresponds to an equal ratio of CFP and YFP fluorescence intensity (result of the above ratio function: 0), red color corresponds to a very low level of YFP fluorescence, while CFP fluorescence levels are still high (result of the above ratio function: approximately 4096).

4.11 Molecular modeling In collaboration with Brian H. Shilton (University of Ontario, London, Canada) a structural analysis of the catalytic domain of PARP10 was performed. The following paragraph gives a short description of this analysis. PARP12 has approximately 33% sequence identity with PARP10, and therefore the recently determined crystal structure of PARP12 (PDB-ID 2PQF; Structural Genomics Consortium, unpublished) supplies a good template for modeling PARP10. To incorporate as much structural information as possible into the PARP10 model and to provide an idea about which regions of the protein can be most reliably modeled, we aligned the structures of human PARP1 (PDB-ID 1UK0 (Kinoshita et al., 2004)) and PARP3 (PDB-ID 2PA9; Structural Genomics Consortium, unpublished) with PARP12. Even though the sequence identity between either PARP1 or PARP3 and PARP12 was less than 20%, there was clear structural similarity: there were 148 CA atoms in PARP12 that were within 3.5 Å of the corresponding CA atoms in PARP1 or PARP3, and the overall RMS distance for these matched positions was 1.4 Å. This structure- based alignment is shown in Figure 2A, where the yellow shading represents structurally conserved regions. Starting with the comprehensive alignment in Otto et al. (Otto et al., 2005), the PARP10 sequence was incorporated into the structure-based Experimental Procedures 126 alignment and submitted to the SwissModel server for threading and optimization (Schwede et al., 2003). There were no serious stereochemical problems with the model that was returned, indicating that the PARP10 sequence was wholly compatible with the template structures. To model the PARP10 binary complex, a “substrate” PARP10 (S-PARP10) was manually positioned onto an “enzymatic” PARP10-NAD+ complex (NAD-PARP10) such that the carboxylate of E882 from S-PARP10 was positioned close to I987 of the NAD- PARP10. In this way, E882 was positioned to act as the catalytic glutamate and can also react with the nicotinamide ribose to form the ADP-ribosylated product. There was only one way to bring the two molecules together such that there were no steric clashes between conserved structural elements and E882 was positioned as described. The complex was energy minimized by manually adjusting two loop regions – namely residues 842-847 and 907-913 – in the NAD-PARP10 molecule, and then making a number of minor side chain and main chain adjustments in both S-PARP10 and NAD-PARP10. This was followed by a round of energy minimization in CNS (Brunger et al., 1998). CA positions were harmonically restrained to maintain the structure of the complex, all other atoms including those of NAD+ were allowed to move. The final PARP10 binary structure had good stereochemistry as assessed by CNS indicators and Procheck (Laskowski et al., 1993) with 83% of residues falling in the most favoured regions of the Ramachandran plot, and no residues in the generously allowed or disallowed regions.

References 127

5. References

Adamietz, P. (1987). Poly(ADP-ribose) synthase is the major endogenous nonhistone acceptor for poly(ADP-ribose) in alkylated rat hepatoma cells. Eur J Biochem 169, 365- 372.

Adamietz, P., Bredehorst, R., and Hilz, H. (1978). Rapid determination of chain length pattern in poly (ADP-ribose) samples. Biochem Biophys Res Commun 81, 1377-1383.

Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., et al. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185-2195.

Aguiar, R. C., Takeyama, K., He, C., Kreinbrink, K., and Shipp, M. A. (2005). B- aggressive lymphoma family proteins have unique domains that modulate transcription and exhibit poly(ADP-ribose) polymerase activity. J Biol Chem 280, 33756-33765.

Aguiar, R. C., Yakushijin, Y., Kharbanda, S., Salgia, R., Fletcher, J. A., and Shipp, M. A. (2000). BAL is a novel risk-related gene in diffuse large B-cell lymphomas that enhances cellular migration. Blood 96, 4328-4334.

Ahel, I., Ahel, D., Matsusaka, T., Clark, A. J., Pines, J., Boulton, S. J., and West, S. C. (2008). Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins. Nature 451, 81-85.

Aktories, K., and Barbieri, J. T. (2005). Bacterial cytotoxins: targeting eukaryotic switches. Nat Rev Microbiol 3, 397-410.

Aktories, K., Jung, M., Bohmer, J., Fritz, G., Vandekerckhove, J., and Just, I. (1995). Studies on the active-site structure of C3-like exoenzymes: involvement of glutamic acid in catalysis of ADP-ribosylation. Biochimie 77, 326-332.

Alkhatib, H. M., Chen, D. F., Cherney, B., Bhatia, K., Notario, V., Giri, C., Stein, G., Slattery, E., Roeder, R. G., and Smulson, M. E. (1987). Cloning and expression of cDNA for human poly(ADP-ribose) polymerase. Proc Natl Acad Sci U S A 84, 1224- 1228.

Allen, T. D., Cronshaw, J. M., Bagley, S., Kiseleva, E., and Goldberg, M. W. (2000). The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. J Cell Sci 113 ( Pt 10), 1651-1659.

Althaus, F. R. (1992). Poly ADP-ribosylation: a histone shuttle mechanism in DNA excision repair. J Cell Sci 102 ( Pt 4), 663-670.

Althaus, F. R., Hofferer, L., Kleczkowska, H. E., Malanga, M., Naegeli, H., Panzeter, P. L., and Realini, C. A. (1994). Histone shuttling by poly ADP-ribosylation. Mol Cell Biochem 138, 53-59.

Althaus, F. R., Kleczkowska, H. E., Malanga, M., Muntener, C. R., Pleschke, J. M., Ebner, M., and Auer, B. (1999). Poly ADP-ribosylation: a DNA break signal mechanism. Mol Cell Biochem 193, 5-11.

References 128

Alvarez-Gonzalez, R. (1988). 3'-Deoxy-NAD+ as a substrate for poly(ADP- ribose)polymerase and the reaction mechanism of poly(ADP-ribose) elongation. J Biol Chem 263, 17690-17696.

Alvarez-Gonzalez, R., and Althaus, F. R. (1989). Poly(ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents. Mutat Res 218, 67-74.

Alvarez-Gonzalez, R., and Jacobson, M. K. (1987). Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo. Biochemistry 26, 3218- 3224.

Ame, J. C., Rolli, V., Schreiber, V., Niedergang, C., Apiou, F., Decker, P., Muller, S., Hoger, T., Menissier-de Murcia, J., and de Murcia, G. (1999). PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J Biol Chem 274, 17860-17868.

Ame, J. C., Spenlehauer, C., and de Murcia, G. (2004). The PARP superfamily. Bioessays 26, 882-893.

Anderson, M. G., Scoggin, K. E., Simbulan-Rosenthal, C. M., and Steadman, J. A. (2000). Identification of poly(ADP-ribose) polymerase as a transcriptional coactivator of the human T-cell leukemia virus type 1 Tax protein. J Virol 74, 2169-2177.

Anderson, P., and Kedersha, N. (2008). Stress granules: the Tao of RNA triage. Trends Biochem Sci 33, 141-150.

Aubin, R. J., Frechette, A., de Murcia, G., Mandel, P., Lord, A., Grondin, G., and Poirier, G. G. (1983). Correlation between endogenous nucleosomal hyper(ADP- ribosyl)ation of histone H1 and the induction of chromatin relaxation. Embo J 2, 1685- 1693.

Audebert, M., and Calsou, P. (2007). Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway. Biochem Biophys Res Commun.

Audebert, M., Salles, B., and Calsou, P. (2004). Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining. J Biol Chem 279, 55117-55126.

Audebert, M., Salles, B., Weinfeld, M., and Calsou, P. (2006). Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double- strand breaks rejoining pathway. J Mol Biol 356, 257-265.

Augustin, A., Spenlehauer, C., Dumond, H., Menissier-De Murcia, J., Piel, M., Schmit, A. C., Apiou, F., Vonesch, J. L., Kock, M., Bornens, M., and De Murcia, G. (2003). PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression. J Cell Sci 116, 1551-1562.

Banasik, M., Komura, H., Shimoyama, M., and Ueda, K. (1992). Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase. J Biol Chem 267, 1569-1575.

Barbieri, J. T., Mende-Mueller, L. M., Rappuoli, R., and Collier, R. J. (1989). Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD. Infect Immun 57, 3549-3554.

References 129

Barford, D., Das, A. K., and Egloff, M. P. (1998). The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27, 133-164.

Bauer, P. I., Buki, K. G., Hakam, A., and Kun, E. (1990). Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity. Biochem J 270, 17-26. Bauer, P. I., Hakam, A., and Kun, E. (1986). Mechanisms of poly(ADP-ribose) polymerase catalysis; mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolar concentrations of NAD. FEBS Lett 195, 331-338.

Bell, C. E., and Eisenberg, D. (1996). Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide. Biochemistry 35, 1137-1149.

Bellocchi, D., Costantino, G., Pellicciari, R., Re, N., Marrone, A., and Coletti, C. (2006). Poly(ADP-ribose)-polymerase-catalyzed hydrolysis of NAD+: QM/MM simulation of the enzyme reaction. ChemMedChem 1, 533-539.

Benchoua, A., Couriaud, C., Guegan, C., Tartier, L., Couvert, P., Friocourt, G., Chelly, J., Menissier-de Murcia, J., and Onteniente, B. (2002). Active caspase-8 translocates into the nucleus of apoptotic cells to inactivate poly(ADP-ribose) polymerase-2. J Biol Chem 277, 34217-34222.

Benjamin, R. C., and Gill, D. M. (1980). Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks. J Biol Chem 255, 10502-10508.

Berger, S. L. (2007). The complex language of chromatin regulation during transcription. Nature 447, 407-412.

Berti, P. J., Blanke, S. R., and Schramm, V. L. (1997). Transition State Structure for the Hydrolysis of NAD+ Catalyzed by Diphtheria Toxin. J Am Chem Soc 119, 12079- 12088.

Bohmer, J., Jung, M., Sehr, P., Fritz, G., Popoff, M., Just, I., and Aktories, K. (1996). Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum-- analysis of glutamic acid 174. Biochemistry 35, 282-289.

Bousset, K., Oelgeschlager, M. H., Henriksson, M., Schreek, S., Burkhardt, H., Litchfield, D. W., Luscher-Firzlaff, J. M., and Luscher, B. (1994). Regulation of transcription factors c-Myc, Max, and c-Myb by casein kinase II. Cell Mol Biol Res 40, 501-511.

Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-921.

Bryant, H. E., Schultz, N., Thomas, H. D., Parker, K. M., Flower, D., Lopez, E., Kyle, S., Meuth, M., Curtin, N. J., and Helleday, T. (2005). Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913-917.

Buki, K. G., and Kun, E. (1988). Polypeptide domains of ADP-ribosyltransferase obtained by digestion with plasmin. Biochemistry 27, 5990-5995.

Burnett, G., and Kennedy, E. P. (1954). The enzymatic phosphorylation of proteins. J Biol Chem 211, 969-980. References 130

Burzio, L. O., Riquelme, P. T., and Koide, S. S. (1979). ADP ribosylation of rat liver nucleosomal core histones. J Biol Chem 254, 3029-3037.

Butler, A. J., and Ordahl, C. P. (1999). Poly(ADP-ribose) polymerase binds with transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific transcription. Mol Cell Biol 19, 296-306.

Caldecott, K. W., Aoufouchi, S., Johnson, P., and Shall, S. (1996). XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular 'nick-sensor' in vitro. Nucleic Acids Res 24, 4387- 4394.

Carroll, S. F., and Collier, R. J. (1984). NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc Natl Acad Sci U S A 81, 3307-3311.

Carroll, S. F., and Collier, R. J. (1987). Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J Biol Chem 262, 8707-8711.

Carroll, S. F., McCloskey, J. A., Crain, P. F., Oppenheimer, N. J., Marschner, T. M., and Collier, R. J. (1985). Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148. Proc Natl Acad Sci U S A 82, 7237-7241. Carter, S. G., and Berger, N. A. (1982). Purification and characterization of human lymphoid poly(adenosine diphosphate ribose) polymerase. Biochemistry 21, 5475- 5481.

Cervellera, M. N., and Sala, A. (2000). Poly(ADP-ribose) polymerase is a B-MYB coactivator. J Biol Chem 275, 10692-10696.

Chadwick, B. P., and Willard, H. F. (2001). Histone H2A variants and the inactive X chromosome: identification of a second macroH2A variant. Hum Mol Genet 10, 1101- 1113.

Chambon, P., Doly, J. D., Strosser, M. T., and Mandel, P. (1966). On the formation of a novel adenylic compound by enzymatic extracts of liver nuclei. Biochem Biophys Res Commun 25, 638-643.

Chambon, P., Weill, J. D., and Mandel, P. (1963). Nicotinamide mononucleotide activation of new DNA-dependent polyadenylic acid synthesizing nuclear enzyme. Biochem Biophys Res Commun 11, 39-43.

Chang, P., Coughlin, M., and Mitchison, T. J. (2005a). Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol 7, 1133- 1139.

Chang, W., Dynek, J. N., and Smith, S. (2005b). NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis. Biochem J 391, 177-184.

Changolkar, L. N., and Pehrson, J. R. (2006). macroH2A1 histone variants are depleted on active genes but concentrated on the inactive X chromosome. Mol Cell Biol 26, 4410-4420.

Chen, C. A., and Okayama, H. (1988). Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632-638. References 131

Cherney, B. W., Chaudhry, B., Bhatia, K., Butt, T. R., and Smulson, M. (1991). Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli. Biochemistry 30, 10420-10427.

Chi, N. W., and Lodish, H. F. (2000). Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J Biol Chem 275, 38437-38444.

Chiang, Y. J., Nguyen, M. L., Gurunathan, S., Kaminker, P., Tessarollo, L., Campisi, J., and Hodes, R. J. (2006). Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice. Mol Cell Biol 26, 2037-2043.

Chou, H. Y., Chou, H. T., and Lee, S. C. (2006). CDK-dependent activation of poly(ADP-ribose) polymerase member 10 (PARP10). J Biol Chem 281, 15201-15207.

Cook, B. D., Dynek, J. N., Chang, W., Shostak, G., and Smith, S. (2002). Role for the related poly(ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol Cell Biol 22, 332-342.

Corda, D., and Di Girolamo, M. (2003). Functional aspects of protein mono-ADP- ribosylation. Embo J 22, 1953-1958.

Costanzi, C., and Pehrson, J. R. (1998). Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393, 599-601.

Costanzi, C., and Pehrson, J. R. (2001). MACROH2A2, a new member of the MARCOH2A core histone family. J Biol Chem 276, 21776-21784.

D'Amours, D., Desnoyers, S., D'Silva, I., and Poirier, G. G. (1999). Poly(ADP- ribosyl)ation reactions in the regulation of nuclear functions. Biochem J 342 ( Pt 2), 249-268.

D'Autreaux, B., and Toledano, M. B. (2007). ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8, 813-824.

Dall'Acqua, W., and Carter, P. (2000). Substrate-assisted catalysis: molecular basis and biological significance. Protein Sci 9, 1-9.

Dantzer, F., Giraud-Panis, M. J., Jaco, I., Ame, J. C., Schultz, I., Blasco, M., Koering, C. E., Gilson, E., Menissier-de Murcia, J., de Murcia, G., and Schreiber, V. (2004). Functional interaction between poly(ADP-Ribose) polymerase 2 (PARP-2) and TRF2: PARP activity negatively regulates TRF2. Mol Cell Biol 24, 1595-1607.

Das, S. K., and Berger, N. A. (1986). Alterations in deoxynucleoside triphosphate metabolism in DNA damaged cells: identification and consequences of poly(ADP- ribose) polymerase dependent and independent processes. Biochem Biophys Res Commun 137, 1153-1158. de Murcia, G., Huletsky, A., Lamarre, D., Gaudreau, A., Pouyet, J., Daune, M., and Poirier, G. G. (1986). Modulation of chromatin superstructure induced by poly(ADP- ribose) synthesis and degradation. J Biol Chem 261, 7011-7017.

Desmarais, Y., Menard, L., Lagueux, J., and Poirier, G. G. (1991). Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity. Biochim Biophys Acta 1078, 179-186.

References 132

Desnoyers, S., Shah, G. M., Brochu, G., Hoflack, J. C., Verreault, A., and Poirier, G. G. (1995). Biochemical properties and function of poly(ADP-ribose) glycohydrolase. Biochimie 77, 433-438.

Doly, J., and Mandel, P. (1967). [Demonstration of the biosynthesis in vivo of a compound polymer, polyadenosine diphosphoribose in the nucleus of the liver of chickens]. C R Acad Sci Hebd Seances Acad Sci D 264, 2687-2690.

Domenighini, M., Magagnoli, C., Pizza, M., and Rappuoli, R. (1994). Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins. Mol Microbiol 14, 41- 50.

Domenighini, M., and Rappuoli, R. (1996). Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages. Mol Microbiol 21, 667-674.

Douglas, C. M., and Collier, R. J. (1990). Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid. Biochemistry 29, 5043-5049.

Doyen, C. M., An, W., Angelov, D., Bondarenko, V., Mietton, F., Studitsky, V. M., Hamiche, A., Roeder, R. G., Bouvet, P., and Dimitrov, S. (2006). Mechanism of polymerase II transcription repression by the histone variant macroH2A. Mol Cell Biol 26, 1156-1164.

Dynek, J. N., and Smith, S. (2004). Resolution of sister telomere association is required for progression through mitosis. Science 304, 97-100.

Edwards, S. L., Brough, R., Lord, C. J., Natrajan, R., Vatcheva, R., Levine, D. A., Boyd, J., Reis-Filho, J. S., and Ashworth, A. (2008). Resistance to therapy caused by intragenic deletion in BRCA2. Nature 451, 1111-1115.

Egloff, M. P., Malet, H., Putics, A., Heinonen, M., Dutartre, H., Frangeul, A., Gruez, A., Campanacci, V., Cambillau, C., Ziebuhr, J., et al. (2006). Structural and functional basis for ADP-ribose and poly(ADP-ribose) binding by viral macro domains. J Virol 80, 8493-8502.

Elliott, G., and Rechsteiner, M. (1975). Pyridine nucleotide metabolism in mitotic cells. J Cell Physiol 86 Suppl 2, 641-651.

Eulalio, A., Behm-Ansmant, I., and Izaurralde, E. (2007). P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Biol 8, 9-22.

Fahrenkrog, B., and Aebi, U. (2003). The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol 4, 757-766.

Farina, K. L., and Singer, R. H. (2002). The nuclear connection in RNA transport and localization. Trends Cell Biol 12, 466-472.

Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N., Johnson, D. A., Richardson, T. B., Santarosa, M., Dillon, K. J., Hickson, I., Knights, C., et al. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917-921.

Fenger-Gron, M., Fillman, C., Norrild, B., and Lykke-Andersen, J. (2005). Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. Mol Cell 20, 905-915. References 133

Gallouzi, I. E., Brennan, C. M., Stenberg, M. G., Swanson, M. S., Eversole, A., Maizels, N., and Steitz, J. A. (2000). HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci U S A 97, 3073-3078.

Gao, G., Guo, X., and Goff, S. P. (2002). Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein. Science 297, 1703-1706.

Gilks, N., Kedersha, N., Ayodele, M., Shen, L., Stoecklin, G., Dember, L. M., and Anderson, P. (2004). Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol Biol Cell 15, 5383-5398.

Gill, D. M., Pappenheimer, A. M., Jr., Brown, R., and Kurnick, J. T. (1969). Studies on the mode of action of diphtheria toxin. VII. Toxin-stimulated hydrolysis of nicotinamide adenine dinucleotide in mammalian cell extracts. J Exp Med 129, 1-21.

Goenka, S., and Boothby, M. (2006). Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor. Proc Natl Acad Sci U S A 103, 4210-4215.

Goenka, S., Cho, S. H., and Boothby, M. (2007). Collaborator of Stat6 (CoaSt6)- associated Poly(ADP-ribose) Polymerase Activity Modulates Stat6-dependent Gene Transcription. J Biol Chem 282, 18732-18739.

Gomez, M., Wu, J., Schreiber, V., Dunlap, J., Dantzer, F., Wang, Y., and Liu, Y. (2006). PARP1 Is a TRF2-associated poly(ADP-ribose)polymerase and protects eroded telomeres. Mol Biol Cell 17, 1686-1696.

Goodwin, P. M., Lewis, P. J., Davies, M. I., Skidmore, C. J., and Shall, S. (1978). The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells. Biochim Biophys Acta 543, 576-582.

Gradwohl, G., Menissier de Murcia, J. M., Molinete, M., Simonin, F., Koken, M., Hoeijmakers, J. H., and de Murcia, G. (1990). The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA. Proc Natl Acad Sci U S A 87, 2990-2994.

Griesenbeck, J., Ziegler, M., Tomilin, N., Schweiger, M., and Oei, S. L. (1999). Stimulation of the catalytic activity of poly(ADP-ribosyl) transferase by transcription factor Yin Yang 1. FEBS Lett 443, 20-24.

Guccione, E., Bassi, C., Casadio, F., Martinato, F., Cesaroni, M., Schuchlautz, H., Luscher, B., and Amati, B. (2007). Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 449, 933-937.

Guccione, E., Martinato, F., Finocchiaro, G., Luzi, L., Tizzoni, L., Dall' Olio, V., Zardo, G., Nervi, C., Bernard, L., and Amati, B. (2006). Myc-binding-site recognition in the human genome is determined by chromatin context. Nat Cell Biol 8, 764-770.

Guiter, C., Dusanter-Fourt, I., Copie-Bergman, C., Boulland, M. L., Le Gouvello, S., Gaulard, P., Leroy, K., and Castellano, F. (2004). Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma. Blood 104, 543-549.

Guo, X., Carroll, J. W., Macdonald, M. R., Goff, S. P., and Gao, G. (2004). The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs. J Virol 78, 12781-12787.

References 134

Guo, X., Ma, J., Sun, J., and Gao, G. (2007). The zinc-finger antiviral protein recruits the RNA processing exosome to degrade the target mRNA. Proc Natl Acad Sci U S A 104, 151-156.

Ha, H. C., Hester, L. D., and Snyder, S. H. (2002). Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia. Proc Natl Acad Sci U S A 99, 3270-3275.

Haigis, M. C., Mostoslavsky, R., Haigis, K. M., Fahie, K., Christodoulou, D. C., Murphy, A. J., Valenzuela, D. M., Yancopoulos, G. D., Karow, M., Blander, G., et al. (2006). SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126, 941-954.

Han, S., and Tainer, J. A. (2002). The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases. Int J Med Microbiol 291, 523-529.

Hanai, S., Uchida, M., Kobayashi, S., Miwa, M., and Uchida, K. (1998). Genomic organization of Drosophila poly(ADP-ribose) polymerase and distribution of its mRNA during development. J Biol Chem 273, 11881-11886.

Harper, J. W., and Adams, P. D. (2001). Cyclin-dependent kinases. Chem Rev 101, 2511-2526.

Hassa, P. O., Buerki, C., Lombardi, C., Imhof, R., and Hottiger, M. O. (2003). Transcriptional coactivation of NF-kappa B-dependent gene expression by p300 is regulated by PARP-1. J Biol Chem.

Hassa, P. O., Haenni, S. S., Buerki, C., Meier, N. I., Lane, W. S., Owen, H., Gersbach, M., Imhof, R., and Hottiger, M. O. (2005). Acetylation of poly(ADP-ribose) polymerase- 1 by p300/CREB-binding protein regulates coactivation of NF-kappaB-dependent transcription. J Biol Chem 280, 40450-40464.

Hassa, P. O., Haenni, S. S., Elser, M., and Hottiger, M. O. (2006). Nuclear ADP- ribosylation reactions in mammalian cells: where are we today and where are we going? Microbiol Mol Biol Rev 70, 789-829.

Hassa, P. O., and Hottiger, M. O. (2002). The functional role of poly(ADP- ribose)polymerase 1 as novel coactivator of NF-kappaB in inflammatory disorders. Cell Mol Life Sci 59, 1534-1553.

Hassa, P. O., and Hottiger, M. O. (2008). The diverse biological roles of mammalian PARPS, a small but powerful family of poly-ADP-ribose polymerases. Front Biosci 13, 3046-3082.

Hayashi, K., Tanaka, M., Shimada, T., Miwa, M., and Sugimura, T. (1983). Size and shape of poly(ADP-ribose): examination by gel filtration, gel electrophoresis and electron microscopy. Biochem Biophys Res Commun 112, 102-107.

Helleday, T., Bryant, H. E., and Schultz, N. (2005). Poly(ADP-ribose) polymerase (PARP-1) in homologous recombination and as a target for cancer therapy. Cell Cycle 4, 1176-1178.

Henriksson, M., and Luscher, B. (1996). Proteins of the Myc network: essential regulators of cell growth and differentiation. Adv Cancer Res 68, 109-182.

References 135

Herceg, Z., and Wang, Z. Q. (2001). Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res 477, 97-110.

Holbourn, K. P., Shone, C. C., and Acharya, K. R. (2006). A family of killer toxins. Exploring the mechanism of ADP-ribosylating toxins. Febs J 273, 4579-4593.

Honjo, T., Nishizuka, Y., and Hayaishi, O. (1968). Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem 243, 3553-3555.

Hsiao, S. J., Poitras, M. F., Cook, B. D., Liu, Y., and Smith, S. (2006). Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping. Mol Cell Biol 26, 2044-2054.

Hsiao, S. J., and Smith, S. (2008). Tankyrase function at telomeres, spindle poles, and beyond. Biochimie 90, 83-92.

Hu, C. D., Chinenov, Y., and Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9, 789-798.

Hunter, T. (2007). The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell 28, 730-738.

Ikejima, M., Marsischky, G., and Gill, D. M. (1987). Direction of elongation of poly(ADP- ribose) chains. Addition of residues at the polymerase-proximal terminus. J Biol Chem 262, 17641-17650.

Ikejima, M., Noguchi, S., Yamashita, R., Ogura, T., Sugimura, T., Gill, D. M., and Miwa, M. (1990). The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA. J Biol Chem 265, 21907-21913.

Ito, S., Shizuta, Y., and Hayaishi, O. (1979). Purification and characterization of poly(ADP-ribose) synthetase from calf thymus. J Biol Chem 254, 3647-3651.

Jagtap, P. G., Baloglu, E., Southan, G. J., Mabley, J. G., Li, H., Zhou, J., van Duzer, J., Salzman, A. L., and Szabo, C. (2005). Discovery of potent poly(ADP-ribose) polymerase-1 inhibitors from the modification of indeno[1,2-c]isoquinolinone. J Med Chem 48, 5100-5103.

Ju, B. G., Lunyak, V. V., Perissi, V., Garcia-Bassets, I., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (2006). A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription. Science 312, 1798-1802.

Ju, B. G., Solum, D., Song, E. J., Lee, K. J., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (2004). Activating the PARP-1 Sensor Component of the Groucho/ TLE1 Corepressor Complex Mediates a CaMKinase IIdelta-Dependent Neurogenic Gene Activation Pathway. Cell 119, 815-829.

Juarez-Salinas, H., Levi, V., Jacobson, E. L., and Jacobson, M. K. (1982). Poly(ADP- ribose) has a branched structure in vivo. J Biol Chem 257, 607-609.

Juszczynski, P., Kutok, J. L., Li, C., Mitra, J., Aguiar, R. C., and Shipp, M. A. (2006). BAL1 and BBAP are regulated by a gamma interferon-responsive bidirectional References 136 promoter and are overexpressed in diffuse large B-cell lymphomas with a prominent inflammatory infiltrate. Mol Cell Biol 26, 5348-5359.

Kameshita, I., Matsuda, M., Nishikimi, M., Ushiro, H., and Shizuta, Y. (1986). Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP- ribose) synthetase. J Biol Chem 261, 3863-3868.

Kameshita, I., Matsuda, Z., Taniguchi, T., and Shizuta, Y. (1984). Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain. J Biol Chem 259, 4770-4776.

Kaminker, P. G., Kim, S. H., Taylor, R. D., Zebarjadian, Y., Funk, W. D., Morin, G. B., Yaswen, P., and Campisi, J. (2001). TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression. J Biol Chem 276, 35891-35899.

Kanai, M., Hanashiro, K., Kim, S. H., Hanai, S., Boulares, A. H., Miwa, M., and Fukasawa, K. (2007). Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation. Nat Cell Biol 9, 1175-1183.

Kanai, Y., Miwa, M., Matsushima, T., and Sugimura, T. (1978). Comparative studies on antibody and antibody production to poly(ADP-ribose) in mice. Immunology 34, 501- 508.

Kannan, P., Yu, Y., Wankhade, S., and Tainsky, M. A. (1999). PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation. Nucleic Acids Res 27, 866-874.

Karras, G. I., Kustatscher, G., Buhecha, H. R., Allen, M. D., Pugieux, C., Sait, F., Bycroft, M., and Ladurner, A. G. (2005). The macro domain is an ADP-ribose binding module. Embo J 24, 1911-1920.

Kaufmann, S. H., Desnoyers, S., Ottaviano, Y., Davidson, N. E., and Poirier, G. G. (1993). Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 53, 3976-3985.

Kawaichi, M., Ueda, K., and Hayaishi, O. (1980). Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase. J Biol Chem 255, 816-819.

Kawaichi, M., Ueda, K., and Hayaishi, O. (1981). Multiple autopoly(ADP-ribosyl)ation of rat liver poly(ADP-ribose) synthetase. Mode of modification and properties of automodified synthetase. J Biol Chem 256, 9483-9489.

Kawamitsu, H., Hoshino, H., Okada, H., Miwa, M., Momoi, H., and Sugimura, T. (1984). Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures. Biochemistry 23, 3771-3777.

Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M. J., Scheuner, D., Kaufman, R. J., Golan, D. E., and Anderson, P. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169, 871-884.

Keith, G., Desgres, J., and de Murcia, G. (1990). Use of two-dimensional thin-layer chromatography for the components study of poly(adenosine diphosphate ribose). Anal Biochem 191, 309-313. References 137

Kerns, J. A., Emerman, M., and Malik, H. S. (2008). Positive selection and increased antiviral activity associated with the PARP-containing isoform of human zinc-finger antiviral protein. PLoS Genet 4, e21.

Khorasanizadeh, S. (2004). The nucleosome: from genomic organization to genomic regulation. Cell 116, 259-272.

Kickhoefer, V. A., Siva, A. C., Kedersha, N. L., Inman, E. M., Ruland, C., Streuli, M., and Rome, L. H. (1999). The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J Cell Biol 146, 917-928.

Kim, H., Jacobson, M. K., Rolli, V., Menissier-de Murcia, J., Reinbolt, J., Simonin, F., Ruf, A., Schulz, G., and de Murcia, G. (1997). Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain. Biochem J 322 ( Pt 2), 469-475.

Kim, M. Y., Mauro, S., Gevry, N., Lis, J. T., and Kraus, W. L. (2004). NAD(+)- Dependent Modulation of Chromatin Structure and Transcription by Nucleosome Binding Properties of PARP-1. Cell 119, 803-814.

Kim, M. Y., Zhang, T., and Kraus, W. L. (2005). Poly(ADP-ribosyl)ation by PARP-1: 'PAR-laying' NAD+ into a nuclear signal. Genes Dev 19, 1951-1967.

Kinoshita, T., Nakanishi, I., Warizaya, M., Iwashita, A., Kido, Y., Hattori, K., and Fujii, T. (2004). Inhibitor-induced structural change of the active site of human poly(ADP-ribose) polymerase. FEBS Lett 556, 43-46.

Kirmizis, A., Santos-Rosa, H., Penkett, C. J., Singer, M. A., Vermeulen, M., Mann, M., Bahler, J., Green, R. D., and Kouzarides, T. (2007). Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation. Nature 449, 928-932.

Kosloff, M., and Selinger, Z. (2001). Substrate assisted catalysis -- application to G proteins. Trends Biochem Sci 26, 161-166.

Kouzarides, T. (2007a). Chromatin modifications and their function. Cell 128, 693-705. Kouzarides, T. (2007b). SnapShot: Histone-Modifying Enzymes. Cell 131, 822.

Kreimeyer, A., Wielckens, K., Adamietz, P., and Hilz, H. (1984). DNA repair-associated ADP-ribosylation in vivo. Modification of histone H1 differs from that of the principal acceptor proteins. J Biol Chem 259, 890-896.

Krishnakumar, R., Gamble, M. J., Frizzell, K. M., Berrocal, J. G., Kininis, M., and Kraus, W. L. (2008). Reciprocal binding of PARP-1 and histone H1 at promoters specifies transcriptional outcomes. Science 319, 819-821.

Kuimov, A. N., Kuprash, D. V., Petrov, V. N., Vdovichenko, K. K., Scanlan, M. J., Jongeneel, C. V., Lagarkova, M. A., and Nedospasov, S. A. (2001). Cloning and characterization of TNKL, a member of tankyrase gene family. Genes Immun 2, 52-55.

Kurosaki, T., Ushiro, H., Mitsuuchi, Y., Suzuki, S., Matsuda, M., Matsuda, Y., Katunuma, N., Kangawa, K., Matsuo, H., Hirose, T., and et al. (1987). Primary structure of human poly(ADP-ribose) synthetase as deduced from cDNA sequence. J Biol Chem 262, 15990-15997.

Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K., and Ladurner, A. G. (2005). Splicing regulates NAD metabolite binding to histone macroH2A. Nat Struct Mol Biol 12, 624-625. References 138

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lagueux, J., Menard, L., Candas, B., Brochu, G., Potvin, F., Verreault, A., Cook, P. F., and Poirier, G. G. (1995). Equilibrium model in an in vitro poly(ADP-ribose) turnover system. Biochim Biophys Acta 1264, 201-208.

Lange, A., Mills, R. E., Lange, C. J., Stewart, M., Devine, S. E., and Corbett, A. H. (2007). Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 282, 5101-5105.

Laskowski, R. A., McArthur, M. W., Moss, D. S., and Thornton, J. M. (1993). PROCHECK: A program to check the stereochemical quality of proteins. J Appl Cryst 26, 283-291.

Le Cam, E., Fack, F., Menissier-de Murcia, J., Cognet, J. A., Barbin, A., Sarantoglou, V., Revet, B., Delain, E., and de Murcia, G. (1994). Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase. J Mol Biol 235, 1062-1071.

Lesniewicz, K., Luscher-Firzlaff, J., Poreba, E., Fuchs, P., Walsemann, G., Wiche, G., and Luscher, B. (2005). Overlap of the gene encoding the novel poly(ADP-ribose) polymerase Parp10 with the plectin 1 gene and common use of exon sequences. Genomics 86, 38-46.

Li, B., Carey, M., and Workman, J. L. (2007). The role of chromatin during transcription. Cell 128, 707-719.

Li, M., Dyda, F., Benhar, I., Pastan, I., and Davies, D. R. (1996). Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation. Proc Natl Acad Sci U S A 93, 6902-6906.

Liszt, G., Ford, E., Kurtev, M., and Guarente, L. (2005). Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J Biol Chem 280, 21313-21320.

Liu, X., Wang, L., Zhao, K., Thompson, P. R., Hwang, Y., Marmorstein, R., and Cole, P. A. (2008). The structural basis of protein acetylation by the p300/CBP transcriptional coactivator. Nature 451, 846-850.

Liu, Y., Snow, B. E., Kickhoefer, V. A., Erdmann, N., Zhou, W., Wakeham, A., Gomez, M., Rome, L. H., and Harrington, L. (2004). Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo. Mol Cell Biol 24, 5314-5323.

Lonskaya, I., Potaman, V. N., Shlyakhtenko, L. S., Oussatcheva, E. A., Lyubchenko, Y. L., and Soldatenkov, V. A. (2005). Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific binding. J Biol Chem 280, 17076-17083.

Lu, X., Nechushtan, H., Ding, F., Rosado, M. F., Singal, R., Alizadeh, A. A., and Lossos, I. S. (2005). Distinct IL-4-induced gene expression, proliferation, and intracellular signaling in germinal center B-cell-like and activated B-cell-like diffuse large-cell lymphomas. Blood 105, 2924-2932.

References 139

Lukac, M., and Collier, R. J. (1988). Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid. J Biol Chem 263, 6146-6149.

Lunde, B. M., Moore, C., and Varani, G. (2007). RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8, 479-490.

Luscher, B., and Eisenman, R. N. (1990). New light on Myc and Myb. Part I. Myc. Genes Dev 4, 2025-2035.

Luscher-Firzlaff, J. M., Lilischkis, R., and Luscher, B. (2006). Regulation of the transcription factor FOXM1c by Cyclin E/CDK2. FEBS Lett 580, 1716-1722.

Lyons, R. J., Deane, R., Lynch, D. K., Ye, Z. S., Sanderson, G. M., Eyre, H. J., Sutherland, G. R., and Daly, R. J. (2001). Identification of a novel human tankyrase through its interaction with the adaptor protein Grb14. J Biol Chem 276, 17172-17180.

Ma, Q. (2001). Induction of CYP1A1. The AhR/DRE paradigm: transcription, receptor regulation, and expanding biological roles. Curr Drug Metab 2, 149-164.

Ma, Q., Baldwin, K. T., Renzelli, A. J., McDaniel, A., and Dong, L. (2001). TCDD- inducible poly(ADP-ribose) polymerase: a novel response to 2,3,7,8- tetrachlorodibenzo-p-dioxin. Biochem Biophys Res Commun 289, 499-506.

Mabley, J. G., Suarez-Pinzon, W. L., Hasko, G., Salzman, A. L., Rabinovitch, A., Kun, E., and Szabo, C. (2001). Inhibition of poly (ADP-ribose) synthetase by gene disruption or inhibition with 5-iodo-6-amino-1,2-benzopyrone protects mice from multiple-low- dose-streptozotocin-induced diabetes. Br J Pharmacol 133, 909-919.

Macara, I. G. (2001). Transport into and out of the nucleus. Microbiol Mol Biol Rev 65, 570-594, table of contents.

Malanga, M., Pleschke, J. M., Kleczkowska, H. E., and Althaus, F. R. (1998). Poly(ADP-ribose) binds to specific domains of p53 and alters its DNA binding functions. J Biol Chem 273, 11839-11843.

Maris, C., Dominguez, C., and Allain, F. H. (2005). The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. Febs J 272, 2118-2131.

Marsischky, G. T., Wilson, B. A., and Collier, R. J. (1995). Role of glutamic acid 988 of human poly-ADP-ribose polymerase in polymer formation. Evidence for active site similarities to the ADP-ribosylating toxins. J Biol Chem 270, 3247-3254.

Matsuo, R., Murayama, A., Saitoh, Y., Sakaki, Y., and Inokuchi, K. (2000). Identification and cataloging of genes induced by long-lasting long-term potentiation in awake rats. J Neurochem 74, 2239-2249.

McDonald, L. J., and Moss, J. (1994). Enzymatic and nonenzymatic ADP-ribosylation of cysteine. Mol Cell Biochem 138, 221-226.

Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (1993). Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular. J Biol Chem 268, 22575-22580.

References 140

Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (1999). Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase. Biochemistry 38, 3948-3953. Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (2001). Regulation of p53 sequence- specific DNA-binding by covalent poly(ADP-ribosyl)ation. J Biol Chem 276, 36425- 36430.

Menissier de Murcia, J., Ricoul, M., Tartier, L., Niedergang, C., Huber, A., Dantzer, F., Schreiber, V., Ame, J. C., Dierich, A., LeMeur, M., et al. (2003). Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse. Embo J 22, 2255-2263.

Menissier-de Murcia, J., Molinete, M., Gradwohl, G., Simonin, F., and de Murcia, G. (1989). Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA. J Mol Biol 210, 229-233.

Meyer, T., and Hilz, H. (1986). Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. Eur J Biochem 155, 157-165.

Meyer, T., Wielckens, K., Thiem, J., and Hilz, H. (1984). NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase. Chemical synthesis and enzymic properties. Eur J Biochem 140, 531-537.

Minaga, T., and Kun, E. (1983a). Probable helical conformation of poly(ADP-ribose). The effect of cations on spectral properties. J Biol Chem 258, 5726-5730.

Minaga, T., and Kun, E. (1983b). Spectral analysis of the conformation of polyadenosine diphosphoribose. Evidence indicating secondary structure. J Biol Chem 258, 725-730.

Miranda, E. A., Dantzer, F., O'Farrell, M., de Murcia, G., and de Murcia, J. M. (1995). Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase. Biochem Biophys Res Commun 212, 317-325.

Miwa, M., Ishihara, M., Takishima, S., Takasuka, N., Maeda, M., Yamaizumi, Z., Sugimura, T., Yokoyama, S., and Miyazawa, T. (1981). The branching and linear portions of poly(adenosine diphosphate ribose) have the same alpha(1 leads to 2) ribose-ribose linkage. J Biol Chem 256, 2916-2921.

Miwa, M., Saikawa, N., Yamaizumi, Z., Nishimura, S., and Sugimura, T. (1979). Structure of poly(adenosine diphosphate ribose): identification of 2'-[1''-ribosyl-2''-(or 3''-)(1'''-ribosyl)]adenosine-5',5'',5'''-tris(phosphate) as a branch linkage. Proc Natl Acad Sci U S A 76, 595-599.

Miwa, M., Saito, H., Sakura, H., Saikawa, N., Watanabe, F., Matsushima, T., and Sugimura, T. (1977). A 13C NMR study of poly(adenosine diphosphate ribose) and its monomers: evidence of alpha-(1'' leads to 2') ribofuranosy1 ribofuranoside risidue. Nucleic Acids Res 4, 3997-4005.

Miyamoto, T., Kakizawa, T., and Hashizume, K. (1999). Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase. Mol Cell Biol 19, 2644-2649.

Muramatsu, Y., Ohishi, T., Sakamoto, M., Tsuruo, T., and Seimiya, H. (2007a). Cross- species difference in telomeric function of tankyrase 1. Cancer Sci 98, 850-857.

References 141

Muramatsu, Y., Tahara, H., Ono, T., Tsuruo, T., and Seimiya, H. (2007b). Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation. Exp Cell Res. Naegeli, H., Loetscher, P., and Althaus, F. R. (1989). Poly ADP-ribosylation of proteins. Processivity of a post-translational modification. J Biol Chem 264, 14382-14385.

Nusinow, D. A., Hernandez-Munoz, I., Fazzio, T. G., Shah, G. M., Kraus, W. L., and Panning, B. (2007). Poly(ADP-ribose) polymerase 1 is inhibited by a histone H2A variant, MacroH2A, and contributes to silencing of the inactive X chromosome. J Biol Chem 282, 12851-12859.

Oei, S. L., and Shi, Y. (2001). Transcription factor Yin Yang 1 stimulates poly(ADP- ribosyl)ation and DNA repair. Biochem Biophys Res Commun 284, 450-454.

Ogata, N., Ueda, K., Kawaichi, M., and Hayaishi, O. (1981). Poly(ADP-ribose) synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei. J Biol Chem 256, 4135-4137.

Ogino, H., Nozaki, T., Gunji, A., Maeda, M., Suzuki, H., Ohta, T., Murakami, Y., Nakagama, H., Sugimura, T., and Masutani, M. (2007). Loss of Parp-1 affects gene expression profile in a genome-wide manner in ES cells and liver cells. BMC Genomics 8, 41.

Oka, J., Ueda, K., Hayaishi, O., Komura, H., and Nakanishi, K. (1984). ADP-ribosyl protein lyase. Purification, properties, and identification of the product. J Biol Chem 259, 986-995.

Oka, S., Kato, J., and Moss, J. (2006). Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase. J Biol Chem 281, 705-713.

Okayama, H., Edson, C. M., Fukushima, M., Ueda, K., and Hayaishi, O. (1977). Purification and properties of poly(adenosine diphosphate ribose) synthetase. J Biol Chem 252, 7000-7005.

Okazaki, I. J., and Moss, J. (1994). Common structure of the catalytic sites of mammalian and bacterial toxin ADP-ribosyltransferases. Mol Cell Biochem 138, 177- 181.

Okazaki, I. J., and Moss, J. (1996). Mono-ADP-ribosylation: a reversible posttranslational modification of proteins. Adv Pharmacol 35, 247-280.

Okazaki, I. J., and Moss, J. (1999). Characterization of glycosylphosphatidylinositiol- anchored, secreted, and intracellular vertebrate mono-ADP-ribosyltransferases. Annu Rev Nutr 19, 485-509.

Oliver, F. J., Menissier-de Murcia, J., Nacci, C., Decker, P., Andriantsitohaina, R., Muller, S., de la Rubia, G., Stoclet, J. C., and de Murcia, G. (1999). Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP- ribose) polymerase-1 deficient mice. Embo J 18, 4446-4454.

Oppenheimer, N. J. (1978). Structural determination and stereospecificity of the choleragen-catalyzed reaction of NAD+ with guanidines. J Biol Chem 253, 4907-4910.

Oppenheimer, N. J., and Bodley, J. W. (1981). Diphtheria toxin. Site and configuration of ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem 256, 8579-8581.

References 142

Otto, H., Reche, P. A., Bazan, F., Dittmar, K., Haag, F., and Koch-Nolte, F. (2005). In silico characterization of the family of PARP-like poly(ADP-ribosyl)transferases (pARTs). BMC Genomics 6, 139.

Ouararhni, K., Hadj-Slimane, R., Ait-Si-Ali, S., Robin, P., Mietton, F., Harel-Bellan, A., Dimitrov, S., and Hamiche, A. (2006). The histone variant mH2A1.1 interferes with transcription by down-regulating PARP-1 enzymatic activity. Genes Dev 20, 3324- 3336.

Pandithage, R., Lilischkis, R., Harting, K., Wolf, A., Jedamzik, B., Luscher-Firzlaff, J., Vervoorts, J., Lasonder, E., Kremmer, E., Knoll, B., and Luscher, B. (2008). The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility. J Cell Biol 180, 915-929.

Panzeter, P. L., and Althaus, F. R. (1990). High resolution size analysis of ADP-ribose polymers using modified DNA sequencing gels. Nucleic Acids Res 18, 2194.

Panzeter, P. L., and Althaus, F. R. (1994). DNA strand break-mediated partitioning of poly(ADP-ribose) polymerase function. Biochemistry 33, 9600-9605.

Parikh, S. L., and Schramm, V. L. (2004). Transition state structure for ADP- ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin. Biochemistry 43, 1204-1212.

Parker, R., and Sheth, U. (2007). P bodies and the control of mRNA translation and degradation. Mol Cell 25, 635-646.

Pavri, R., Lewis, B., Kim, T. K., Dilworth, F. J., Erdjument-Bromage, H., Tempst, P., de Murcia, G., Evans, R., Chambon, P., and Reinberg, D. (2005). PARP-1 determines specificity in a retinoid signaling pathway via direct modulation of mediator. Mol Cell 18, 83-96.

Pawson, T., Gish, G. D., and Nash, P. (2001). SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 11, 504-511.

Pehrson, J. R., Costanzi, C., and Dharia, C. (1997). Developmental and tissue expression patterns of histone macroH2A1 subtypes. J Cell Biochem 65, 107-113.

Pemberton, L. F., and Paschal, B. M. (2005). Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187-198.

Petrucco, S., and Percudani, R. (2008). Structural recognition of DNA by poly(ADP- ribose)polymerase-like zinc finger families. Febs J.

Petzold, S. J., Booth, B. A., Leimbach, G. A., and Berger, N. A. (1981). Purification and properties of poly(ADP-ribose) polymerase from lamb thymus. Biochemistry 20, 7075- 7081.

Pizza, M., Bartoloni, A., Prugnola, A., Silvestri, S., and Rappuoli, R. (1988). Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity. Proc Natl Acad Sci U S A 85, 7521-7525.

Plafker, K., and Macara, I. G. (2000). Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1. Mol Cell Biol 20, 3510-3521.

References 143

Plaza, S., Aumercier, M., Bailly, M., Dozier, C., and Saule, S. (1999). Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina. Oncogene 18, 1041-1051.

Pleschke, J. M., Kleczkowska, H. E., Strohm, M., and Althaus, F. R. (2000). Poly(ADP- ribose) binds to specific domains in DNA damage checkpoint proteins. J Biol Chem 275, 40974-40980.

Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982). Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc Natl Acad Sci U S A 79, 3423-3427.

Prigent, C., and Dimitrov, S. (2003). Phosphorylation of serine 10 in histone H3, what for? J Cell Sci 116, 3677-3685.

Quimby, B. B., and Dasso, M. (2003). The small GTPase Ran: interpreting the signs. Curr Opin Cell Biol 15, 338-344.

Ratnam, K., and Low, J. A. (2007). Current development of clinical inhibitors of poly(ADP-ribose) polymerase in oncology. Clin Cancer Res 13, 1383-1388.

Raval-Fernandes, S., Kickhoefer, V. A., Kitchen, C., and Rome, L. H. (2005). Increased susceptibility of vault poly(ADP-ribose) polymerase-deficient mice to carcinogen- induced tumorigenesis. Cancer Res 65, 8846-8852.

Realini, C. A., and Althaus, F. R. (1992). Histone shuttling by poly(ADP-ribosylation). J Biol Chem 267, 18858-18865.

Rechsteiner, M., Hillyard, D., and Olivera, B. M. (1976). Turnover at nicotinamide adenine dinucleotide in cultures of human cells. J Cell Physiol 88, 207-217.

Reeder, R. H., Ueda, K., Honjo, T., Nishizuka, Y., and Hayaishi, O. (1967). Studies on the polymer of adenosine diphosphate ribose. II. Characterization of the polymer. J Biol Chem 242, 3172-3179.

Ribbeck, K., and Gorlich, D. (2002). The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. Embo J 21, 2664-2671.

Rippmann, J. F., Damm, K., and Schnapp, A. (2002). Functional characterization of the poly(ADP-ribose) polymerase activity of tankyrase 1, a potential regulator of telomere length. J Mol Biol 323, 217-224.

Riquelme, P. T., Burzio, L. O., and Koide, S. S. (1979). ADP ribosylation of rat liver lysine-rich histone in vitro. J Biol Chem 254, 3018-3028.

Rising, K. A., and Schramm, V. L. (1997). Transition State Analysis of NAD+ Hydrolysis by the Cholera Toxin Catalytic Subunit. J Am Chem Soc 119, 27-37.

Rolli, V., O'Farrell, M., Menissier-de Murcia, J., and de Murcia, G. (1997). Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching. Biochemistry 36, 12147-12154.

Rouleau, M., McDonald, D., Gagne, P., Ouellet, M. E., Droit, A., Hunter, J. M., Dutertre, S., Prigent, C., Hendzel, M. J., and Poirier, G. G. (2007). PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery. J Cell Biochem 100, 385-401. References 144

Ruf, A., de Murcia, G., and Schulz, G. E. (1998a). Inhibitor and NAD+ binding to poly(ADP-ribose) polymerase as derived from crystal structures and homology modeling. Biochemistry 37, 3893-3900.

Ruf, A., Mennissier de Murcia, J., de Murcia, G., and Schulz, G. E. (1996). Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken. Proc Natl Acad Sci U S A 93, 7481-7485.

Ruf, A., Rolli, V., de Murcia, G., and Schulz, G. E. (1998b). The mechanism of the elongation and branching reaction of poly(ADP-ribose) polymerase as derived from crystal structures and mutagenesis. J Mol Biol 278, 57-65.

Saito, I., Hatakeyama, K., Kido, T., Ohkubo, H., Nakanishi, S., and Ueda, K. (1990). Cloning of a full-length cDNA encoding bovine thymus poly(ADP-ribose) synthetase: evolutionarily conserved segments and their potential functions. Gene 90, 249-254.

Sakai, W., Swisher, E. M., Karlan, B. Y., Agarwal, M. K., Higgins, J., Friedman, C., Villegas, E., Jacquemont, C., Farrugia, D. J., Couch, F. J., et al. (2008). Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers. Nature 451, 1116-1120.

Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., et al. (2008). Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487-498.

Sambrook, J., Fritsch, E. F., Maniatis, T., and (eds.) (1989). Molecular Cloning - A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, New York.

Sastry, S. S., Buki, K. G., and Kun, E. (1989). Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs. Biochemistry 28, 5670-5680.

Sauve, A. A., Wolberger, C., Schramm, V. L., and Boeke, J. D. (2006). The biochemistry of sirtuins. Annu Rev Biochem 75, 435-465.

Saxena, A., Wong, L. H., Kalitsis, P., Earle, E., Shaffer, L. G., and Choo, K. H. (2002). Poly(ADP-ribose) polymerase 2 localizes to mammalian active centromeres and interacts with PARP-1, Cenpa, Cenpb and Bub3, but not Cenpc. Hum Mol Genet 11, 2319-2329.

Sbodio, J. I., and Chi, N. W. (2002). Identification of a tankyrase-binding motif shared by IRAP, TAB182, and human TRF1 but not mouse TRF1. NuMA contains this RXXPDG motif and is a novel tankyrase partner. J Biol Chem 277, 31887-31892.

Sbodio, J. I., Lodish, H. F., and Chi, N. W. (2002). Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1 (telomere-repeat-binding factor 1) and IRAP (insulin-responsive aminopeptidase). Biochem J 361, 451-459.

Scheuring, J., Berti, P. J., and Schramm, V. L. (1998). Transition-state structure for the ADP-ribosylation of recombinant Gialpha1 subunits by pertussis toxin. Biochemistry 37, 2748-2758.

Scheuring, J., and Schramm, V. L. (1995). Stereochemistry of the ADP-Ribosylation Catalyzed by Pertussis Toxin. J Am Chem Soc 117, 12653-12654.

References 145

Scheuring, J., and Schramm, V. L. (1997). Kinetic isotope effect characterization of the transition state for oxidized nicotinamide adenine dinucleotide hydrolysis by pertussis toxin. Biochemistry 36, 4526-4534.

Schreiber, V., Ame, J. C., Dolle, P., Schultz, I., Rinaldi, B., Fraulob, V., Menissier-de Murcia, J., and de Murcia, G. (2002). Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1. J Biol Chem 277, 23028-23036.

Schreiber, V., Dantzer, F., Ame, J. C., and de Murcia, G. (2006). Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 7, 517-528.

Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003). SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31, 3381-3385.

Seimiya, H., Muramatsu, Y., Smith, S., and Tsuruo, T. (2004). Functional subdomain in the ankyrin domain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation. Mol Cell Biol 24, 1944-1955.

Seman, M., Adriouch, S., Haag, F., and Koch-Nolte, F. (2004). Ecto-ADP- ribosyltransferases (ARTs): emerging actors in cell communication and signaling. Curr Med Chem 11, 857-872.

Sessler, R. J., and Noy, N. (2005). A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II. Mol Cell 18, 343-353.

Sibley, J. T., Braun, R. P., and Lee, J. S. (1986). The production of antibodies to DNA in normal mice following immunization with poly(ADP-ribose). Clin Exp Immunol 64, 563-569.

Sibley, J. T., Latimer, L. J., and Lee, J. S. (1988). Shared idiotypes on anti-DNA and anti-poly (ADP-ribose) antibodies. J Immunol 140, 3502-3507.

Simbulan-Rosenthal, C. M., Ly, D. H., Rosenthal, D. S., Konopka, G., Luo, R., Wang, Z. Q., Schultz, P. G., and Smulson, M. E. (2000). Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase. Proc Natl Acad Sci U S A 97, 11274-11279.

Simbulan-Rosenthal, C. M., Rosenthal, D. S., Luo, R., Samara, R., Espinoza, L. A., Hassa, P. O., Hottiger, M. O., and Smulson, M. E. (2003). PARP-1 binds E2F-1 independently of its DNA binding and catalytic domains, and acts as a novel coactivator of E2F-1-mediated transcription during re-entry of quiescent cells into S phase. Oncogene 22, 8460-8471.

Simonin, F., Hofferer, L., Panzeter, P. L., Muller, S., de Murcia, G., and Althaus, F. R. (1993a). The carboxyl-terminal domain of human poly(ADP-ribose) polymerase. Overproduction in Escherichia coli, large scale purification, and characterization. J Biol Chem 268, 13454-13461.

Simonin, F., Menissier-de Murcia, J., Poch, O., Muller, S., Gradwohl, G., Molinete, M., Penning, C., Keith, G., and de Murcia, G. (1990). Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity. J Biol Chem 265, 19249-19256.

References 146

Simonin, F., Poch, O., Delarue, M., and de Murcia, G. (1993b). Identification of potential active-site residues in the human poly(ADP-ribose) polymerase. J Biol Chem 268, 8529-8535.

Sims, J. L., Berger, S. J., and Berger, N. A. (1983). Poly(ADP-ribose) Polymerase inhibitors preserve nicotinamide adenine dinucleotide and adenosine 5'-triphosphate pools in DNA-damaged cells: mechanism of stimulation of unscheduled DNA synthesis. Biochemistry 22, 5188-5194.

Singh, N., Poirier, G., and Cerutti, P. (1985). Tumor promoter phorbol-12-myristate-13- acetate induces poly ADP-ribosylation in human monocytes. Biochem Biophys Res Commun 126, 1208-1214.

Skidmore, C. J., Davies, M. I., Goodwin, P. M., Halldorsson, H., Lewis, P. J., Shall, S., and Zia'ee, A. A. (1979). The involvement of poly(ADP-ribose) polymerase in the degradation of NAD caused by gamma-radiation and N-methyl-N-nitrosourea. Eur J Biochem 101, 135-142.

Smets, L. A., Loesberg, C., Janssen, M., and Van Rooij, H. (1990). Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis. Biochim Biophys Acta 1054, 49-55.

Smith, S., and de Lange, T. (1999). Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes. J Cell Sci 112 ( Pt 21), 3649-3656.

Smith, S., and de Lange, T. (2000). Tankyrase promotes telomere elongation in human cells. Curr Biol 10, 1299-1302.

Smith, S., Giriat, I., Schmitt, A., and de Lange, T. (1998). Tankyrase, a poly(ADP- ribose) polymerase at human telomeres. Science 282, 1484-1487.

Soldatenkov, V. A., Chasovskikh, S., Potaman, V. N., Trofimova, I., Smulson, M. E., and Dritschilo, A. (2002). Transcriptional repression by binding of poly(ADP-ribose) polymerase to promoter sequences. J Biol Chem 277, 665-670.

Southan, G. J., and Szabo, C. (2003). Poly(ADP-ribose) polymerase inhibitors. Curr Med Chem 10, 321-340.

Spector, D. L. (2006). SnapShot: Cellular bodies. Cell 127, 1071.

Stewart, M. (2007). Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8, 195-208.

Stoecklin, G., and Anderson, P. (2007). In a tight spot: ARE-mRNAs at processing bodies. Genes Dev 21, 627-631.

Strahl, B. D., and Allis, C. D. (2000). The language of covalent histone modifications. Nature 403, 41-45.

Sugimura, T., Fujimura, S., Hasegawa, S., and Kawamura, Y. (1967). Polymerization of the adenosine 5'-diphosphate ribose moiety of NAD by rat liver nuclear enzyme. Biochim Biophys Acta 138, 438-441.

References 147

Sun, J., Maresso, A. W., Kim, J. J., and Barbieri, J. T. (2004). How bacterial ADP- ribosylating toxins recognize substrates. Nat Struct Mol Biol 11, 868-876.

Takada, T., Iida, K., and Moss, J. (1993). Cloning and site-directed mutagenesis of human ADP-ribosylarginine hydrolase. J Biol Chem 268, 17837-17843.

Takeyama, K., Aguiar, R. C., Gu, L., He, C., Freeman, G. J., Kutok, J. L., Aster, J. C., and Shipp, M. A. (2003). The BAL-binding protein BBAP and related Deltex family members exhibit ubiquitin-protein isopeptide ligase activity. J Biol Chem 278, 21930- 21937.

Tanaka, M., Hayashi, K., Sakura, H., Miwa, M., Matsushima, T., and Sugimura, T. (1978). Demonstration of high molecular weight poly (adenosine diphosphate ribose). Nucleic Acids Res 5, 3183-3194.

Terry, L. J., Shows, E. B., and Wente, S. R. (2007). Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318, 1412-1416.

Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995). Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81, 801-809.

Thibodeau, J., Potvin, F., Kirkland, J. B., and Poirier, G. (1993). Expression in Escherichia coli of the 36 kDa domain of poly(ADP-ribose) polymerase and investigation of its DNA binding properties. Biochim Biophys Acta 1163, 49-53.

Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350-4354.

Tulin, A., and Spradling, A. (2003). Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299, 560-562.

Tulin, A., Stewart, D., and Spradling, A. C. (2002). The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev 16, 2108-2119.

Uchida, K., Hanai, S., Ishikawa, K., Ozawa, Y., Uchida, M., Sugimura, T., and Miwa, M. (1993). Cloning of cDNA encoding Drosophila poly(ADP-ribose) polymerase: leucine zipper in the auto-modification domain. Proc Natl Acad Sci U S A 90, 3481-3485.

Uchida, K., Morita, T., Sato, T., Ogura, T., Yamashita, R., Noguchi, S., Suzuki, H., Nyunoya, H., Miwa, M., and Sugimura, T. (1987). Nucleotide sequence of a full-length cDNA for human fibroblast poly(ADP-ribose) polymerase. Biochem Biophys Res Commun 148, 617-622.

Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979). Poly(ADP-ribosy)ation of nuclear proteins. Enzymatic elongation of chemically synthesized ADP-ribose- histone adducts. J Biol Chem 254, 679-687.

Venkitaraman, A. R. (2002). Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108, 171-182.

Vermeulen, M., Mulder, K. W., Denissov, S., Pijnappel, W. W., van Schaik, F. M., Varier, R. A., Baltissen, M. P., Stunnenberg, H. G., Mann, M., and Timmers, H. T. References 148

(2007). Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58-69.

Wacker, D. A., Ruhl, D. D., Balagamwala, E. H., Hope, K. M., Zhang, T., and Kraus, W. L. (2007). The DNA binding and catalytic domains of poly(ADP-ribose) polymerase 1 cooperate in the regulation of chromatin structure and transcription. Mol Cell Biol 27, 7475-7485.

Wang, Z. Q., Auer, B., Stingl, L., Berghammer, H., Haidacher, D., Schweiger, M., and Wagner, E. F. (1995). Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev 9, 509-520.

Wielckens, K., George, E., Pless, T., and Hilz, H. (1983). Stimulation of poly(ADP- ribosyl)ation during Ehrlich ascites tumor cell "starvation" and suppression of concomitant DNA fragmentation by benzamide. J Biol Chem 258, 4098-4104.

Williams, G. T., Lau, K. M., Coote, J. M., and Johnstone, A. P. (1985). NAD metabolism and mitogen stimulation of human lymphocytes. Exp Cell Res 160, 419-426.

Woon, E. C., and Threadgill, M. D. (2005). Poly(ADP-ribose)polymerase inhibition - where now? Curr Med Chem 12, 2373-2392.

Yamasaki, S., Stoecklin, G., Kedersha, N., Simarro, M., and Anderson, P. (2007). T- cell intracellular antigen-1 (TIA-1)-induced translational silencing promotes the decay of selected mRNAs. J Biol Chem 282, 30070-30077.

Yan, K. S., Kuti, M., and Zhou, M. M. (2002). PTB or not PTB -- that is the question. FEBS Lett 513, 67-70.

Ye, J. Z., and de Lange, T. (2004). TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex. Nat Genet 36, 618-623.

Yeh, T. Y., Sbodio, J. I., and Chi, N. W. (2006). Mitotic phosphorylation of tankyrase, a PARP that promotes spindle assembly, by GSK3. Biochem Biophys Res Commun 350, 574-579.

Yeh, T. Y., Sbodio, J. I., Nguyen, M. T., Meyer, T. N., Lee, R. M., and Chi, N. W. (2005). Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1. Mol Cell Biochem 276, 183-192.

Ying, W. (2006). NAD+ and NADH in cellular functions and cell death. Front Biosci 11, 3129-3148.

Yu, J. H., Yang, W. H., Gulick, T., Bloch, K. D., and Bloch, D. B. (2005a). Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body. Rna 11, 1795-1802.

Yu, M., Schreek, S., Cerni, C., Schamberger, C., Lesniewicz, K., Poreba, E., Vervoorts, J., Walsemann, G., Grötzinger, J., Kremmer, E., et al. (2005b). PARP-10, a novel Myc- interacting protein with poly(ADP-ribose) polymerase activity, inhibits transformation. Oncogene 24, 1982-1993.

Zahradka, P., and Ebisuzaki, K. (1982). A shuttle mechanism for DNA-protein interactions. The regulation of poly(ADP-ribose) polymerase. Eur J Biochem 127, 579- 585.

References 149

Zhang, Y., Burke, C. W., Ryman, K. D., and Klimstra, W. B. (2007). Identification and characterization of interferon-induced proteins that inhibit alphavirus replication. J Virol 81, 11246-11255.

Zheng, C. L., Sumizawa, T., Che, X. F., Tsuyama, S., Furukawa, T., Haraguchi, M., Gao, H., Gotanda, T., Jueng, H. C., Murata, F., and Akiyama, S. (2005). Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes. Biochem Biophys Res Commun 326, 100-107.

Appendix 150

6. Appendix

6.1 Abbreviations °C Degree Celsius A Ampere aa Amino acid Ab Antibody Ad5 Adenovirus 5 A.dest. Destilled water ADP Adenosine diphosphate Amp Ampicillin AMP Adenosine monophosphate APS Ammonium persulfate ARC Ankyrin repeat cluster ARH ADP-Ribose hydrolase ART ADP-Ribosyltransferase ATCC American Type Culture Collection ATP Adenosine triphosphate ß-ME ß-Mercaptoethanol BAL B-Aggressive Lymphoma BER Base excision repair bHLH Basic helix-loop-helix bp base pair(s) BPB Bromophenole blue Bq Bequerel BSA Bovine serum albumin ca. circa CA C alpha CaM Calmodulin CBB Coomassie brilliant blue CFP Cyan fluorescent protein CDK Cyclin-dependent kinase ChIP Chromatin immunoprecipitation Ci Curie cm2 square centimeter CMV Cytomegalie virus Da Dalton Appendix 151

DBD DNA binding domain DLBCL Diffuse large B cell lymphoma DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid DRE Dioxin response elements DSB Double strand break DTT Dithiothreitol ECL Enhanced chemoluminescens E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid e.g. for example EGTA Ethyleneglycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid ES Cell Embryonic stem cell EST Expressed sequence tag et.al. et alii f.a.v. Final assay volume FCCP Carbonyl cyanide p-trifluoromethoxyphenylhydrazone FCS Fetal calf serum FISH Fluorescence In Situ Hybridisation g Gramm g gravitation constant ( g = 9,81 m / s) GAP GTPase activating protein GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GSK Glycogen synthase kinase GTP Guanosine triphosphate h hour(s) Hebs Hepes buffered saline HEK Human embryo kidney HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HMG High mobility group HMT Histone methyltransferase HRP Horseradish peroxidase i.e. that is IgG Immunoglobulin G IP Immunoprecipitation Appendix 152

IPTG Isopropyl-ß-D-1-thiogalacopyranoside IRAP Insulin-responsive amino peptidase ITC Isothermal calorimetry k Kilo KLH Keyhole limpet hemocyanin wave length of light l Litre LB Luria Broth LOH Loss of heterozygosity LPS Lipopolysaccharide Micro m Meter m Milli M Mega M Molar (mol / l) mART Mono-ADP-Ribosyltransferase MCS Multiple cloning site MDCK Madin-Darby canine kidney MIBG meta-Iodobenzylguanidine min Minute(s) MNNG N-Methyl-N'-Nitro-N-Nitrosoguanidine mRNA messenger RNA MS Mass spectrometry n Nano NAD Nicotinamide adenindinucleotide NEB New England Biolabs NES Nuclear export signal NHEJ Non-homologous end joining NLS Nuclear localisation signal NP-40 Nonidet P-40 NPC Nuclear pore complex NuMA Nuclear mitotic apparatus protein o- ortho- OAADPR O-Acetyl-ADP-ribose OD Optical density p.a. pro analysi PAGE Polyacrylamide gel electrophoresis Appendix 153

PAR Poly-ADP-ribose pADPr Poly-ADP-ribose PARG Poly-ADP-ribose glycohydrolase PARP Poly-ADP-ribose polymerase PBS Phospate buffered saline PBZ Poly-ADP-ribose-binding zinc finger PcG Polycomb group PKR Protein kinase R PMSF Phenylmethylsulphonyl fluoride PTM Post-translational modification PVDF Polyvinylidene fluoride QM/MM Quantum mechanical / Molecular mechanical RAR Retinoic acid receptor REF Rat embryo fibroblast RNA Ribonucleic acid RPMI Roswell Park Memorial Institute RRM RNA recognition motif rpm rounds per minute SAC Substrate-assisted catalysis SDS Sodium dodecyl sulfate shRNA short hairpin RNA SSB Single strand break TAP Tandem affinity purification TB Terrific Broth TCA Trichloroacetic acid TCDD 2,3,7,8-Tetrachlorodibenzo-p-dioxin TE Tris-EDTA TEP Telomere-associated protein TEV Tobacco Etch virus U Unit UIM Ubiquitin-interacting motif v/v volume per volume Vol. Volume w/v weight per volume WB Western Blot WT Wild-type x times / -fold Appendix 154

XC Xylene cyanol YFP Yellow fluorescent protein

Appendix 155

6.2 Curriculum vitae

Name: Schuchlautz Vorname: Henning Geburtsdatum: 1.12.1979 Geburtsort: Waldbröl Nationalität: Deutsch Familienstand: ledig Konfession: römisch-katholisch

Schulbildung: 1986-1990 Besuch der Grundschule „Sonnenschule“ in Attendorn 1990-1999 Besuch des Rivius-Gymnasiums in Attendorn Juni 1999 Erlangung der Allgemeinen Hochschulreife

Studium: 1999-2005 Diplom-Studiengang Biologie an der Rheinisch- Westfälischen Technischen Hochschule (RWTH) Aachen 09/2004-06/2005 Diplomarbeit am Institut für Biochemie der RWTH Aachen, Abtlg. Biochemie und Molekularbiologie, AG Prof. B. Lüscher Juni 2005 Erlangung des Grades Diplom-Biologe

Berufstätigkeit: seit August 2005 im Rahmen einer Promotion Mitarbeiter am Institut für Biochemie der RWTH Aachen, Abtlg. Biochemie und Molekularbiologie, AG Prof. B. Lüscher Appendix 156

6.3 Veröffentlichungen Publikationen in wissenschaftlichen Journalen: Guccione, E., Bassi, C., Casadio, F., Martinato, F., Cesaroni, M., Schuchlautz, H., Lüscher, B., and Amati. B. (2007). Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 449: 933-38.0

Lüscher-Firzlaff, J., Gawlista, I., Vervoorts, J., Kapelle, K., Braunschweig, T., Walsemann, G., Rodgarkia-Schamberger, C., Schuchlautz, H., Dreschers, S., Kremmer, E., Lilischkis, R., Cerni, C., Wellmann, A., and Lüscher B. (2008). The human trithorax protein hASH2 functions as an oncoprotein. Cancer Res 68(3): 749-58.

Schuchlautz, H., Poreba, E., Lesniewicz, K., Hassa, P.O., Hottiger, M.O., Litchfield, D.W., Shilton, B.H., and Lüscher, B. (2008). Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation. In Revision

Vorträge auf wissenschaftlichen Konferenzen: Schuchlautz, H., Yu, M., Lüscher-Firzlaff, J., Montzka, K., Grötzinger, J., Lesniewicz, K., Poreba, E., Litchfield, D.W., and Lüscher, B. (2005). Characterization and function of PARP10 in the control of cell behavior. Talk given at: 16th International Symposium on Poly-ADP-ribosylation, PARP2005: Bench to bedside, October 5-7, Gateshead, UK.

Schuchlautz, H., Lesniewsicz, K., Poreba, E., and Lüscher, B. (2006). PARP10, a non- classical PARP enzyme that affects cell proliferation. Talk given at: 3rd PARP Regio Meeting, May 19, Illkirch, France.

Schuchlautz, H., Lesniewicz, K., Poreba, E., and Lüscher, B. (2007). PARP10: PARP meets mART. Talk given at: 4th PARP Regio Meeting, September 3-4, EMBL, Heidelberg, Germany.

Posterpräsentationen auf wissenschaftlichen Konferenzen: Schuchlautz, H., Stöcklin, G., Herrmann, A., Müller-Newen, G., Lüscher-Firzlaff, J., and Lüscher. B. (2008). Analysis of PARP10’s nucleocytoplasmic shuttling and identification of PARP10 foci. Poster presented at: 17th International Symposium on Poly-ADP-ribosylation, May 28-31, Tucson, Arizona, USA.

Appendix 157

6.3 Eidesstattliche Erklärung

Ich erkläre eidesstattlich, dass ich die vorliegende Dissertation selbständig verfasst und alle in Anspruch genommenen Hilfen in der Dissertation angegeben habe. Des Weiteren erkläre ich, dass die vorliegende Dissertation nicht bereits als Diplomarbeit oder vergleichbare Prüfungsarbeit verwendet worden ist.

Aachen, den 15.4.2008 Henning Schuchlautz

Appendix 158

6.4 Danksagung Danken möchte ich zuerst Prof. Dr. Bernhard Lüscher für die Möglichkeit zur Promotion auf diesem interessanten Themengebiet, sein Vertrauen in meine Arbeit, die stetige Unterstützung und Förderung in den letzten Jahren, viele wissenschaftliche Diskussionen und Anregungen und alles, was ich im Rahmen meiner Promotion von ihm lernen durfte.

PD Dr. Christoph Peterhänsel danke ich für die Übernahme des Referats.

Dr. Paul O. Hassa, Dr. Andreas Herrmann, Prof. Dr. Michael Hottiger, Prof. Dr. Gerhard Müller-Newen, Dr. Antonio S. Sechi, Dr. Georg Stöcklin, Michael Vogt und insbesondere Prof. Dr. Brian H. Shilton bin ich zu Dank verpflichtet wegen ihrer stetigen Mithilfe an diesem Projekt, welches durch ihre Kooperationsbereitschaft um zahlreiche Aspekte bereichert wurde.

Für die gute Atmosphäre beim Arbeiten, wissenschaftlichen Austausch und vieles mehr bedanke ich mich bei den jetzigen und ehemaligen Mitgliedern der AG Lüscher: Dr. Poornima Basavarajaiah, Kathrin Borggrebe, Anne Braczynski, Stefan Brüning, Elena Buerova, Heike Chauvistre, Christian Cornelissen, Dr. Stefan Dreschers, Isabella Gawlista, Andrea Graf, Nadine Hein, Britta Jedamzik, Dr. Kan Jiang, Karsten Kapelle, Angelina Kriescher, Dr. Richard Lilischkis, Ulrike Linzen, Dr. Juliane Lüscher-Firzlaff, Gabriele Lützeler, Larissa Milke, Elke Meyer, Katrin Montzka, Alexandra Neuss, Dr. Ruwin Pandithage, Krischen Ray, Marcel Robbertz, Jens Schirrmacher, Anne Schröder, Vera Schumacher, Mikola Seeliger, Stefanie Speckgens, Jürgen Stahl, Angelika Szameit, Susanne Waibel, Dr. Gesa Walsemann, Alexandra Wolf, Dr. Jörg Vervoorts-Weber, Dr. Mei Yu.

Meinen Freunden aus dem Sauerland, besonders meinen Schulfreunden vom Knobelclub A.H.B. und Vereinskameraden vom Spielmannszug Biekhofen, und meinen Freunden in Aachen, besonders Katrin und Phong, danke ich für viele frohe Stunden und willkommene Abwechslung von den „Mühen der Wissenschaft“. Weiter so!

Ich danke meinen Eltern und auch meinen zukünftigen Schwiegereltern sowie den gesamten Familien Tandler und Kleine für ihre kontinuierliche Unterstützung in den letzten Jahren. Gut, dass es euch gibt!

Zu guter Letzt ein großer Dank an Katrin – sie weiß wofür!