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Dissertation Published Version Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 Biochemical and regulatory aspects of DNA double-strand break repair Godau, Julia Eileen Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-170542 Dissertation Published Version Originally published at: Godau, Julia Eileen. Biochemical and regulatory aspects of DNA double-strand break repair. 2018, University of Zurich, Faculty of Science. Biochemical and Regulatory Aspects of DNA Double-Strand Break Repair Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Julia Eileen Godau aus Deutschland Promotionskommission Prof. Dr. Alessandro A. Sartori (Vorsitz und Leitung der Dissertation) Prof. Dr. Joao Matos PD. Dr. Pavel Janscak Prof. Dr. Matthias Altmeyer Zürich, 2018 Contents Abbreviations v Summary vii Zusammenfassung ix 1 Introduction 1 1.1 Genome instability - A hallmark of cancer . 1 1.2 DNA damage response . 2 1.3 DNA repair . 3 1.3.1 DSB repair . 4 1.3.2 Meiotic recombination . 6 1.3.3 Fanconi anemia pathway of ICL repair . 7 1.4 Post-translational modifications . 10 1.5 CtIP . 11 1.5.1 Sae2/Ctp1/CtIP protein family . 11 1.5.2 CtIP promotes DNA-end resection . 13 1.5.3 Regulation of CtIP by PTMs . 15 1.5.4 CtIP and its connection to cancer development and therapy . 18 1.6 SLX4 - A nuclease scaffold . 19 1.7 PIN1 . 22 1.7.1 PIN1 - A molecular switch regulating diverse pathways . 22 1.7.2 PIN1 and its role in tumorigenesis . 25 1.8 P. tetraurelia as a model organism to study DSB repair . 27 2 Aims 31 3 Material and Methods 33 4 Results 41 4.1 Investigating the biochemical function of CtIP in DSB repair . 41 4.1.1 Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair . 41 Supplementary Figures . 79 i 4.1.2 Paramecium CtIP stimulates MRE11 nuclease activity . 85 4.2 Deciphering the potential regulation of SLX4 by PIN1- mediated isomerization . 89 4.2.1 Validation of the PIN1-SLX4 interaction . 89 4.2.2 Identification of the PIN1-interaction motif in SLX4 . 89 4.2.3 Establishing a phospho-SLX4 (T1315) antibody . 97 4.2.4 Investigating the potential role of PIN1 in regulating SLX4 function . 98 5 Discussion and Outlook 109 5.1 Investigating the biochemical function of CtIP in DSB repair . 109 5.2 Deciphering the potential regulation of SLX4 by PIN1- mediated isomerization . 115 Bibliography 119 Acknowledgments 147 Appendix 149 Curriculum Vitae . 150 Publication: A Short BRCA2-Derived Cell-Penetrating Peptide Targets RAD51 Function and Confers Hypersensitivity toward PARP Inhibition. 152 ii Abbreviations ALT alternative lengthening of telomeres AML acute myeloid leukemia Aph aphidicolin ATM ataxia-telangiectasia mutated ATR ataxia-telangiectasia and Rad3-related BER base excision repair CDC25 cell division cycle 25 family members CDK cyclin-dependent kinase CFS common fragile sites CO crossover CPT camptothecin CTD C-terminal domain CtIP CtBP-interacting protein DDR DNA damage response dHJ double Holliday junction DNA deoxyribonucleic acid DNA-PKcs DNA-dependent protein kinase catalytic subunit ds double-stranded DSB double-strand break Ess1 essential in yeast 1 FA fanconi anemia FAAP24 FA-associated protein 24 kDa GSK3 glycogen synthase kinase 3 HJ Holliday junction HR homologous recombination ICL interstrand crosslink IES internal eliminated sequences IP immunoprecipitation IR Ionizing radiation JNK Jun-N-terminal protein kinase LE long exposure MAC macronucleus MAPK mitogen-activated protein kinases MEF mouse embryonic fibroblasts MIC micronucleus MMC mitomycin C MMR mismatch repair v MRE11 meiotic recombination 11 homolog A MRN MRE11-RAD50-NBS1 NBS1 nijmegen breakage syndrome 1 NER nucleotide excision repair NHEJ non-homologous end joining NIMA never in mitosis A NTD N-terminal domain PARP poly(ADP-ribose) polymerase PD pulldown PIKK phosphatidylinositol 3-kinase-like protein kinase PLK1 polo-like kinase 1 PPase protein phosphatase PPIase peptidyl-prolyl cis/trans isomerase PTMs posttranslational modifications RB retinoblastoma RBBP8 retinoblastoma binding protein 8 Rtt107 regulator of Ty1 transposition ROS reactive-oxygen species RPA replication protein A Sae2 sporulation in the absence of Spo11 protein 2 homolog SC synaptonemal complex SE short exposure SIM SUMO interaction motif SLX synthetic lethal of unknown function Spo11 sporulation-specific protein 11 ss single-stranded SSB single-strand breaks ssc single-stranded circular S/T-P serine/threonine-proline SUMO small ubiquitin-related modifiers TLS translesion synthesis UV ultraviolet light wt wild type vi Summary Faithful transmission of genetic information to daughter cells is fundamental for all living cells. DNA lesions resulting from exogenous or endogenous sources are potent driving forces of genomic instability and tumorigenesis. To maintain genome integrity, cells have evolved a complex network of DNA damage signaling and repair pathways, referred to as the DNA damage response (DDR). CtIP and SLX4 are key DDR factors, contributing to the error-free survival of cells in response to genotoxic stress. However, our mechanistic understanding about how these DNA repair proteins operate at the molecular level and how their activities are regulated during the cell cycle is very limited. Human CtIP is crucial for the resection and repair of DNA double-strand breaks (DSBs), one of the most cytotoxic lesions a cell can encounter. At the molecular level, the func- tion of CtIP in DSB repair is not fully understood and the lack of high-resolution structures precludes any detailed insights into its biochemical features. Recently, our lab identified two open reading frames in the ciliate Paramecium tetraurelia, representing the short- est known orthologs of CtIP in evolution. In the course of our studies, we investigated the DNA binding properties of recombinant PtCtIP and its functional interplay with the MRE11-RAD50 nuclease complex. Specifically, we find that PtCtIP binds with high affin- ity to replication fork-like DNA structures but lacks any detectable intrinsic nuclease ac- tivity. Moreover, DNA binding, but not MRE11-RAD50 interaction, strongly depends on a highly conserved RHR motif located in the evolutionarily conserved C-terminus. Addi- tionally, using genetically engineered cells stably expressing a CtIP DNA binding mutant, we provide evidence that the RHR motif mediates recruitment of CtIP to sites of DNA damage, thereby ensuring efficient DNA-end resection and homology-directed repair of DSBs. In collaboration with the lab of Dr. Mireille Bétermier, we further unveiled that vii PtCtIP is required for the processing of Spo11-dependent meiotic DSBs in P. tetraurelia. Together, these results establish that PtCtIP is a true homolog of CtIP and that a short DNA-interaction motif is crucial for its function in DSB repair. In the second part of my Ph.D. thesis, we sought to decipher the potential regulation of SLX4 by PIN1-mediated prolyl isomerization. PIN1 catalyzes the isomerization of pro- line peptide bounds adjacent to phosphorylated serines or threonines (pS/T-P) to either cis or trans configuration, thereby controlling protein function. We previously discovered various DDR factors in a proteomic screen for potential PIN1 targets, including the DNA repair endonuclease scaffold SLX4. Human SLX4 is involved in the cleavage of DNA interstrand-crosslinks (ICL) and resolution of Holliday junctions (HJs) as well as in the trimming of telomeric loops. Our results hitherto suggest that PIN1 binds to SLX4 in a direct and phosphorylation-dependent manner. Moreover, fine-mapping of the PIN1 interaction motif in SLX4 points towards multiple pS/T-P sites as critical sites for PIN1 recognition. However, overexpression or knockdown of PIN1 does neither alter estab- lished SLX4 protein-protein interactions required for ICL repair and HJ resolution nor its telomeric localization, indicating that PIN1 potentially controls other biological aspects involving SLX4. viii Zusammenfassung Jeden Tag wird unser Erbgut - DNS genannt - geschädigt, sei es durch UV-Strahlung, Tabakrauch oder andere toxische Substanzen, die bei normalen zellinneren Prozessen entstehen. Jede Veränderung der DNS birgt die Gefahr entscheidende Mechanismen, welche die Zellteilung und das Zellwachstum kontrollieren, ausser Gefecht zu setzen. Dies kann zu Zellentartung und schliesslich Krebs führen. Umso entscheidender ist, dass jede einzelne Zelle mit einem vielfältigen Repertoire an DNS Reparaturmechanismen ausgestattet ist, welche die Integrität des Genoms schützen. Das Zusammenspiel einzelner Faktoren in diesem hoch komplexen Netzwerk - unter ih- nen Schlüsselproteine wie CtIP und SLX4 - ist jedoch bei weitem nicht aufgeklärt. CtIP ist ein evolutionär hochkonserviertes Protein, welches eine essentielle Rolle in der Reparatur von DNS Doppelstrangbrüchen (DSB) spielt. Auch wenn in den letzten Jahren viele Erkenntnisse über CtIP gewonnen wurden, fehlen immer noch entscheidende Mo- saikstücke um die Funktion von CtIP im Detail zu verstehen. Erschwerend kommt hinzu, dass die Kristallstruktur von CtIP noch nicht aufgeklärt werden konnte. Diese würde uns wichtige Anhaltspunkte zum besseren Verständnis der biochemischen Eigenschaften von CtIP liefern. Der erste Teil meiner Arbeit beschreibt die Charakterisierung von CtIP des Pantoffeltierchens
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