DUE-B in CHROMATIN and NUCLEAR SPECKLES a Thesis

DUE-B IN CHROMATIN AND NUCLEAR SPECKLES

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science

NADIA KATRANGI M.D., Damascus University, 1993

2007 Wright State University

NADIA KATRANGI

2007

WRIGHT STATE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

May 23, 2007

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Nadia Katrangi ENTITLED DUE- B in Chromatin and Nuclear Speckles BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science .

Michael Leffak, Ph.D. Thesis Director

Daniel Organisciak, Ph.D.

Department Chair Committee on Final Examination

Steve Berberich, Ph.D.

Paula Bubulya, Ph.D.

Madhavi Kadakia, Ph.D.

Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies

ABSTRACT

Katrangi, Nadia. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2007. DUE-B in chromatin and Nuclear Speckles.

The DNA unwinding element binding protein (DUE-B) was first identified by using a yeast one hybrid screen with the DNA unwinding element (DUE) from the c-myc origin as bait. DUE-B’s orthologue in the yeast Saccharomyces cerevisiae lacks the last 60 C-terminal amino acids and has been identified as a D-tyrosyl-tRNA deacylase. A substantial group of evidence suggests a role for DUE-B in the regulation of replication initiation. Here we show that DUE-B is focused in nuclear speckles and colocalizes with spliceosome associated protein 145 (SAP145), an mRNA splicing factor 3B subunit. Mass spectrometry results show that SAP145 co-purifies with the

6xHis-tagged DUE-B. Surprisingly, ∆CT- DUE-B, the DUE-B mutant lacking the 60 C-terminal amino acids, appeared almost exclusively in nuclear speckles whereas its yeast orthologue was found to be cytoplasmic. DUE-B’s distribution pattern did not change in cells arrested in G1/S and it did not appear in replication foci despite the strong evidence of its involvement in replication initiation. Finally, DUE-B’s proposed interaction with DNA methyltransferase 1

(Dnmt1) is further investigated by immunofluorescence. Interestingly, results show that this interaction with Dnmt1 seems to occur in nuclear speckles. Based on DUE-B’s interacting proteins and its concentration in nuclear speckles, a possible role in the DNA damage response is discussed.

TABLE OF CONTENTS

Page

I. INTRODUCTION AND PURPOSE ...... 1

II. MATERIALS AND METHODS ...... 7

Cell culture ...... 7

Transient transfection ...... 7

Immunofluorescence...... 7

DUE-B purification assay...... 10

Western blot analysis ...... 10

Mass spectrometry analysis sample preparation ...... 11

Cell fractionation ...... 12

Gel filtration assay ...... 13

III. RESULTS ...... 14

DUE-B is localized in nuclear speckles in HeLa cells ...... 14

SAP145 co-purifies with His-tagged DUE-B ...... 17

DUE-B colocalizes with SAP145 in speckles ...... 19

C-Terminus deleted DUE-B mutant is more concentrated in nuclear speckles than the Wild-Type ...... 19

DUE-B does not appear in replication sites by immunofluorescence or change its speckled pattern at late G1/early S...... 22

DUE-B in the nucleus is on DNA and in nuclear speckles ...... 24

DUE-B colocalizes partially with Dnmt1 in speckles ...... 27

DUE-B does not colocalize with PML-NB ...... 31

IV. Discussion ...... 33

V. References ...... 38

LIST OF FIGURES

Figure Page

1. DUE-B crystal structre ...... 2

2. DUE-B’s amino acid sequence ...... 3

3. Nuclear compartments...... 4

4. Immunofluorescence protocol ...... 8

5. Mass spectrometry preparation procedure ...... 11

6. DUE-B is in speckles in HeLa and A1 cells ...... 14

7. Mcm3 displays a different distribution pattern than DUE-B...... 15

8. Speckled pattern with the His-antibody is specific to DUE-B ...... 16

9. WT DUE-B colocalization with hSm proteins...... 17

10. Mass spectrometry results ...... 18

11. WT DUE-B co-elutes with SAP145 ...... 19

12. WT DUE-B colocalizes with SAP145 ...... 19

13. ∆CT DUE-B in speckles ...... 20

14. DUE-B colocalizes in speckles with SAP145 and hSm ...... 21

15. ∆CT DUE-B is more enriched in speckles than the WT DUE-B ...... 22

16. DUE-B in G1 and S phases ...... 23

17. DUE-B loses concentration in speckles during mitosis...... 24

18. The cell fractionation procedure ...... 25

19. DUE-B in chromatin fraction is sensitive to Dnase I treatment and contains speckled proteins. . . .26

20. DUE-B interacts with Dnmt1...... 27

21. Dnmt1 is seen at some replication foci in A1 cells . . .28

22. DUE-B partially colocalizes with Dnmt1...... 29

23. Dnmt1 appears in speckles in some cells ...... 30

24. Dnmt1 appears in speckles in HeLa cells,A1 and ∆CT cells labeled only with anti-Dnmt1 ...... 31

25.DUE-B does not colocalize with PML-NB ...... 32

vii ACKNOWLEDGEMENT

I wish to express my gratitude to my advisor Dr. Michael Leffak for his mentoring and support. He provided a great educational environment. I would also like to thank Dr. Paula Bubulya for providing me with resources and the use of her equipment. I truly appreciate all her help and advice. I am grateful to all the members in Dr. Leffak’s lab. The spirit of teamwork in the lab allowed me to grow and progress in the program. Dr. Michael Kemp’s work was the basis of my project and I am particularly thankful for his continuous support and help throughout my thesis project.

viii Introduction

DNA replication, one of the main nuclear functions in non-quiescent cells, initiates at origins of replication upon the sequential binding/release of the replication proteins to or from these origins (Machida et al., 2005; Bell and Dutta, 2002). A common characteristic of eukaryotic origins of replication is the presence of an area of helical instability known as the DNA unwinding element (DUE) (Liu et al., 2003; Natale at al., 1993). Upstream of the c-myc gene a

2.4 kb fragment was identified as an origin of replication (Malott and Leffak, 1999; McWhinney et al., 1995; McWhinney and Leffak, 1990; Leffak and James, 1989; Trivedi et al., 1998). This origin contained a DUE (Bazar et al., 1995) as well as three matches to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus sequence (Malott and Leffak,

1999). The c-myc origin activity was eliminated upon the deletion of the ARS/DUE region (Liu et al., 2003), thus, implying the requirement of DUE for origin activity. To identify proteins that might regulate the c-myc origin, a yeast one hybrid assay was used with the DUE from the c-myc origin as bait. A 23.4 kD protein was found and was named the DNA unwinding element binding protein (DUE-B) (Casper et al., 2005). Human DUE-B (hDUE-B) has 209 amino acids. It is isolated as a homodimer (Figure 1) and it is conserved in eukaryotes (Figure 2). The yeast S. cerevisiae orthologue lacks ~ 60 amino acids in the carboxy-terminus and was recently identified as a D-tyrosyl-tRNA deacylase (Soutourina et al., 2000). DUE-B purified with ATPase activity and was proposed to be involved in regulating replication initiation. This suggestion was based on chromatin immunoprecipitation assay results, which showed DUE-B to be bound near the c-myc replicator DUE in a cell cycle dependent manner (Casper et al., 2005; Ghosh et al., 2006). DUE-

B also appeared to be preferentially phosphorylated in cells arrested in early S phase.

Additionally, RNA interference for DUE-B delayed entry into S phase and promoted cell death in

HeLa cells (Casper et al., 2005). Most significantly, immunodepletion of DUE-B in Xenopus egg extracts inhibited DNA replication and the addition of recombinant DUE-B, purified from HeLa

1 cells, rescued the replication activity (Casper et al., 2005). The crystal structure of the first 151 amino acids (Figure 1), on the other hand, showed a remarkable similarity to the structure of both

D-tyrosyl tRNA deacylase (Ferri-Fioni at al., 2001, 2006) and the archeal-specific editing domain of threonyl-tRNA synthetase (Hussain et al., 2006). DUE-B, however, appears to have an Mg²+ ion in the active site (Figure 1) (Kemp et al., 2007), which was not observed in any of the tRNA editing enzymes.

hDUE-B D-Tyr-tRNA Deacylase Hemophilus influenzae

Figure 1: DUE -B Crystal Structure. A, DUE-B crystal structure resembles that of D-tyr-tRNA deacylase. B, DUE-B monomer including the magnesium ion co-crystallized with each DUE-B monomer. (Kemp et al., 2007)

Recently, DUE-B has been suggested to interact with DNA methyltransferase 1 (Dnmt1)

(Kemp and Leffak, unpublished data) whose main function is to maintain the methylation pattern after DNA replication (Bestor, 2000). A fraction of Dnmt1 is a constituent of a multiprotein DNA replication complex (Vertino et al., 2002). Interestingly, Dnmt1 has been suggested to be necessary for the activity of several origins including the c -myc origin (Knox et al., 2000). Dnmt1

2 also has a role in restoring the epigenetic information during DNA repair (Mortusewicz et al.,

2005).

hDUE -B /fulllength MKAVVQRVTRASVTVGGEQISAIGRGICVLLGISLEDTQKELEHMVR KILNL RVFED -ESGKHWSKSVMDKQYEILCVSQFTLQCVL K-GN KPDFHLAMPTEQAEGFYNSFLEQL 113 hDUE -B / ∆ CT MKAVVQRVTRASVTVGGEQISAIGRGICVLLGISLEDTQKELEHMVR KILNL RVFED-ESGKHWSKSVMDKQYEILCVSQFTLQCVL K-GN KPDFHLAMPTEQAEGFYNSFLEQL 113

hDUE -B /fulllength RKTYRPELIKDGKFGAYMQVHIQNDGPVTIELESPAPGTATSDPKQLSKLEKQQQRKEKTRAKGPSESSKERNTPR -KEDRS ASSGAEGDVSSEREP GTGHHHHHH hDUE -B / ∆ CT RKTYRPELIKDGKFGAYMQVHIQNDGPVTIELES GTGHHHHHH His-tag A1 DUE-B His-tag ∆ CT DUE-B

Human MKAVVQRVTRASVTVGGEQISAIGRGICVLLGISLEDTQKELEHMVR KILNL RVFED-ESGKHWSKSVMDKQYEILCVSQFTLQCVL K-GN KPDFHLAMPTEQAEGFYNSFLEQL 113 Mouse MKAVVQRVTRASVTVGGEQISAIGRGICVLLGISMEDSQKELEHMVR KILNL RVFED-ESGKHWSKSVMDKEYEVLCVSQFTLQCVL K-GN KPDFHLAMPTEQAESFYNSFLEQL 113 Chicken MKAIVQRVAQASVTVGGEQISSIGRGLCVLLGISLEDTQRELEHMVR KILNL RVFED-ESGKHWSKSVMDKQYEVLCVSQFTLQCIL K-GN KPDYHMAMPTEQAESFYNNFLEQL 113 Frog MRAVIQRVTKASVTVGDEQISSIGRGICVLLGISVEDTQKDIEYMVR KILNL RLFTD-ESGKPWCKSVMDKQYEVLCVSQFTLQCVL K-GN KPDYHMAMPSEQAEPFYNNFLQHM 113 Zebrafish MKAIIQRVTRASVTVGEEQISSIGRGLCVLLGISAEDTQKDVDYMVR KILNL RVFED-ENGRAWSRSVMDGELEVLCVSQFTLQCLL K-GN KPDYHAAMPAELAQPFYNNMLEQL 113 Pufferfish MKAVVQRVVRASVCVGDEQVSSIGRGLCVLLGISAEDTQSDADYMIRKILNLRLFAD-ENGRAWSKSVMDLDYEVLCVSQFTLQCMLK-GNKPDFHAAMPAELAQPFYSHILENM 113 Worm MRVVLQRVTRAAVTVSDEVVGSIGRGLCVLVGIHRDDTEEDMKYIIR KILNL RIFPA-SEEKPWDKSVMDLDLEVLSVSQFTLYGQF K-GN KLDFHTAMAPTEASKFYATFLESL 113 Fruitfly MRAVIQRVKAAKVTVLDELVSSIGPGLCVLVGIKASDTAKDVEYLVR KILAL RLFEE--EGKRWQKSVKDLNLELLCVSQFTLYHRL K-GN KPDFLAAMKGEEAQELYNQFLDRL 112 Arabidopsis MRAVIQRVSSSSVTVDGRIVSEIGPGLLVLIGIHESDTESDADYICR KVLNM RLFSNETTGKGWDQNVMQRNYGVLLVSQFTLYGFL K-GN KPDFHVAMPPDKAKPFYASLVEKF 114 Fission Yeast MKAVIQRVLNASVSVDDKIVSAIQQGYCILLGVGSDDTPEDVTKLSN KILKL KLFDN--AEQPWKSTIADIQGEILCVSQFTLHARVN KGA KPDFHRSMKGPEAIELYEQVVKTL 113 Budding Yeast MKIVLQKVSQASVVVDSKVISSIKHGYMLLVGISIDDSMAEIDKLSK KVLSL RIFED-ESRNLWKKNIKEANGEILSVSQFTLMAKTK KGT KPDFHLAQKGHIAKELYEEFLKLL 114 E. coli MIALIQRVTRASVTVEGEVTGEIGAGLLVLLGVEKDDDEQKANRLCE RVLGY RIFSD--AEGKMNLNVQQAGGSVLVVSQFTLAADTE RGM RPSFSKGASPDRAEALYDYFVERC 113 Hemophilus MIALIQRVSQAKVDVKGETIGKIGKGLLVLLGVEKEDNREKADKLAE KVLNY RIFSD--ENDKMNLNVQQAQGELLIVSQFTLAADTQ KGL RPSFSKGASPALANELYEYFIQKC 113

Human RKTYRPELIKDGKFGAYMQVHIQNDGPVTIELESPAPGTATSDPKQLSKLEKQQQRKEKTRAKGPSESSKERNTPR-KEDRSASSGAEGDVSSEREP 209 Mouse RKSYRPELIRDGKFGAYMQVHIQNDGPVTIELESPAPGAASSDPKQLSKLEKQQQRKEKTRAKGPSESSKERNAPR-KEDRSASSGAEGDVSSEREP 209 Chicken RKAYKPELIKDGKFGAYMQVHIQNDGPVTIELESPA---AAVDPKQLAKLEKQQQRKEKTRTKVPSESSRERNAPRSKDDPSASSGAEGDVSSEREP 207 Frog RKAYKPELIKDGKFGAYMQLNIQNDGPVTIELEPPA---STADPKLLSKLEKQQQRKEKTRTKTQSESSREKSVPRSKDDPSASSGAEGDVSSEREP 207 Zebrafish RETYKPELIKDGQFGAKMQVLIQNDGPVTIQLESPP---APTDPKLLSKQEKQQQRKEKTRSKGPSDSSREKAAQRSKVDPSASSGAEGDVSSEREP 207 Pufferfish RSIYKPEHIKDGKFGAKMQVNIQNDGPVTIELTSPS---APTDPKLISKQEKQQQRKEKTRSKGPSESSK-KSGQWHRREPNTSSGAEGDVSSEKE- 205 Worm KKAYKPEKIQDGKFAAMMSVDIVNDGPVTISFDSKEK------Fruitfly GQSYDSTKIK-----AYMQVHIENDGPVTINLESPEQKDTDREVDK------Arabidopsis QKAYNPDAVKDGVFGAMMQVNLVNDGPVTMQLDSPQSTK------Fission Yeast GESLGSDKIKKGVFGAMMNVQLVNNGPVTILYDTKE------Budding Yeast RSDLGEEKVKDGEFGAMMSCSLTNEGPVTIILDSDQ------E. coli RQQE--MNTQTGRFAADMQVSLVNDGPVTFWLQV------Hemophilus AEK---LPVSTGQFAADMQVSLTNDGPVTFWLNV------

DUE -B amino acid sequence Figure 2: : DUE-B is conserved in other eukaryotes [mouse (93%) and frog (>80%) identical]. DUE-B’s orthologue in lower eukaryotes is identified as D- tyr-tRNA deacylase and has ~147 amino acids whereas human DUE-B is 209. amino acids.

The processes of transcription, apoptosis, replication and repair are integrated through the involvement of specific proteins in multiple pathways (Aravind and Koonin, 2000; Li and Manely,

2006). Proteins of defined roles are now found to contribute in other nuclear functions. For example, studying the interaction between the proliferating cell nuclear antigen (PCNA), which has a well recognized role as a sliding clamp for loading DNA replication and repair proteins on

DNA, and the transcription co-activator p300 has lead to proposing a role for PCNA in transcription regulation (Hong and Chakravarti, 2003) and for p300 in DNA repair (Hasan et al.,

2001). Another example are the aminoacyl-tRNA synthetases (ARS) and their interacting factors

(ARS interacting multifunctional proteins, AIMPs), which have recently been shown to

3 participate in various functions including transcription, splicing, DNA replication and repair

(Park et al., 2005).

Figure 3: Nuclear Compartments (Kosak and Groudine, 2004)

Understanding cellular structure has been a key-stone in revealing the functions of the cellular organelles as well as those of their protein contents . The nuclear architecture in eukaryotic cells is based on chromatin arrangement where chromatin occupies distinct regions while the areas void of chromatin, known as the interchromatin compartment, contain different substructures (Figure

3). Factors for transcription, splicing, DNA replication and repair reside in these substructures

(Cremer & Cremer, 2001; Kosak and Groudine, 2004). The most prominent difference between the cytoplasmic and nuclear structures of a eukaryotic cell is that cytoplasmic compartments are sequestered by membranes, whereas their nuclear counterparts are not (Carrero et al., 2005). Yet, the nucleus is highly organized and compartmentalized while still very dynamic (Dundr, 2001;

Lamond and Earnshaw, 1998; Lamond and Spector, 2003). With the use of electron and

4 immunofluorescence microscopes (Spector, 1993; Platasani et al., 2000) and biochemical extraction of the nuclear organelles (Mintz, et al., 1999; Saitoh et al., 2004; Schirmer, 2003 ), some organelles were found to have clearly distinguished functions and well characterized protein components. Nucleoli, for example, were identified as the sites of ribosomal transcription, modification and pre-ribosome assembly (Handwerger and Gall, 2006). Cajal bodies are believed to participate in the processing of ribosomal and messenger RNA (Gall, 2000). Lately, however, increasing evidence is suggesting that some nuclear compartments might be contributing to nuclear functions besides their original recognized ones such as promyelocytic leukemia nuclear bodies (PML-NB) and the interchromatin granular clusters (IGCs). PML-NBs were suggested to be involved in several functions including protein degradation (Lallemand-Breitenbach et al.,

2001), apoptosis (Wang et al., 1998), tumor suppression and transcription regulation (Seeler and

Dejean, 1999). More studies are now predicting a role for PML-NBs in DNA repair (Dellaire et al., 2004). IGCs, which became a model for understanding functional compartmentalization in the nucleus, are commonly accepted as the place of assembly, modification and storage for splicing factors (Lamond and Spector, 2003). However, they are now found to contain proteins involved in apoptosis and DNA repair (Saitoh et al., 2004) and predicted to have unexpected new roles, particularly, in cellular stress responses (Fayolle et al., 2006; Campalans et al., 2007).

IGCs are also called nuclear speckles due to their distribution pattern in the interchromatin compartment. There are 25-50 speckles in the nucleus each measuring ~1.0-1.5 µm and containing clusters of ~25 nm granules. Although the majority of IGCs’ components are related to mRNA maturation, no single motif common to all IGCs’ proteins has been identified (Saitoh et al., 2004). The RS motif, serine-arginine or arginine-serine dipeptide repeats, is the most prominent among many splicing factors (SF) and speckle proteins such as SF2 and SC35. Other domains were detected in a small subset of protein-speckled components like the SAP motif that is found in spliceosome associated protein 145 (SAP145), a subunit of the SF3 (Aravind and

Koonin, 2000; Schmidt-Zachmann et al., 1998). The SAP motif is named after SAF-A/B, acinus

5 and PIAS (Aravind and Koonin, 2000) and is thought to be present in proteins that are associated with both chromatin and RNA (Saitoh et al., 2004).

The current study demonstrates yet another new example of the proposed regulatory cross- talk among the various nuclear functions. Shown here by immunofluorescence, DUE-B is predominantly observed in nuclear speckles where it colocalizes with SAP145. Also shown,

DUE-B co-purifies with SAP145 according to mass spectrometry results. Additionally, DUE-B’s previous suggested interaction with Dnmt1 is confirmed here by their colocalization, which, unexpectedly, appears to be in nuclear speckles as well. Immunofluorescence and chromatin fractionation results together suggest that DUE-B appears in at least two nuclear compartments: loosely or tightly bound to chromatin and localized in nuclear speckles.

6 Materials and Methods

Cell Culture

The cells used were HeLa, A1 and ∆CT cell lines. The latter two cell lines are HeLa cells stably over-expressing full-length DUE-B in A1 and expressing DUE-B lacking the last 62 amino acids in ∆CT. In both A1 and ∆CT cell lines, exogenous DUE-B has a 6xHis- tag at its C- terminus. Cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% new born calf serum and gentamicin (50 µg/ml) (Gibco) at 37˚C in 5% CO 2.

Transient Transfection

Plasmid pcDNA 3.1D/V5-His/ lac Z (Invitrogen), previously cloned in E. coli, was extracted from the bacteria and purified by using a QIAprep Spin Miniprep Kit and protocol (Qiagen). The amount of plasmid purified was estimated by running a sample on an agarose gel. HeLa cells were grown 24 hours prior to transfection without antibiotics. Cells were 95% confluent at the time of transfection. LacZ plasmid was transiently transfected to HeLa cells using Lipofectamine

2000 (Invitrogen) according to the method provided by the manufacturer. Six hours after transfection, cells were plated on cover slips for immunofluorescence experiments to be performed 30-36 hours later.

Immunofluorescence

Cells were grown 36-48 hours prior to experiment on glass cover slips. At the time of the experiment, cells would be 30-50% confluent. Cells were washed with phosphate buffered saline

(PBS) then fixed with 2% paraformaldehyde, freshly dissolved in PBS, for 15 minutes at room temperature then washed with PBS for 3 times, 5 minutes each. In most experiments, cells were extracted prior to fixation by incubating them for 2 minutes on ice with 0.2% Triton X-100 in a buffer containing 10 mM HEPES, 50 mM KCl 2, 25 mM MgCl 2 and 200 mM sucrose. After

7 fixation, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes at room temperature then washed with 0.5% newborn goat serum (NGS) in PBS (blocking buffer) 3 times,

5 minutes each. Cells were incubated with the primary antibodies diluted (antibodies and their dilutions are described below) in a volume of 30-50 µl of the blocking buffer in a humid chamber for one hour, washed 3 times, 5 minutes each with the blocking buffer then incubated with the secondary antibodies for one hour in the dark. Afterward, cells on the cover slips were washed 3 times, 5 minutes each. DAPI (Sigma) was added in the last wash for DNA staining; cells were then mounted for viewing on the microscope. The procedure is summarized in Figure 4 (The mounting medium and the procedure were obtained from Dr. P. Bubulya, Wright State

University).

Pre-extraction 0.2% Triton X-100 2 min/ice

Fixation 2% paraformaldehyde 15 min/room temperature

Permeabilization 0.2 Triton X-100 5 min/room temperature

Block 0.5% NGS 3x5 min

Primary antibody 1hr

Wash with blocking buffer 3x5 min

Figure 4: Secondary antibody 1hr Immunofluorescence protocol. Pre-extraction Wash with blocking buffer 3x5 min/DAPI was not applied in all

immunofluorescence mount experiments

8 For bromodeoxyuridine (BrdU) incorporation and DNA synthesis labeling, cells were incubated with 10 µM BrdU for 20 minutes at 37˚C prior to starting the experiment, washed with

PBS, pre-extracted, fixed then permeabilized as mentioned above. Ten units of RNase free DNase

I (Takara Bio Inc.) and anti-BrdU were added with the primary antibodies for 2 hours followed by washing 3 times, 5 minutes each, incubation for 1 hour with secondary antibodies, washing then mounting as stated previously.

In case of UV treatment to induce DNA damage, cells on cover slips were placed in PBS, irradiated with 60 J/m² UV dose in a UV crosslinker (Stratagene), then re-incubated in the culture medium at 37˚C for 2 hours. Following the two hour incubation, the immunofluorescence experiment was performed as above.

For cell synchronization, aphidicolin and mimosine were used at final concentrations of 1

µg/ml and 0.2 mM respectively. Cells were incubated with either of the two drugs for 18 hours prior to the immunofluorescence experiment.

The following primary antibodies were used for immunofluorescence: anti-DUE-B rabbit polyclonal antibody, generated as described previously (Casper et al., 2005), at a dilution of 1:5, anti-His mouse monoclonal antibody (Invitrogen) at a dilution of 1:200 (or 1:10 in some of the experiments performed on ∆CT cells), anti-PML rabbit polyclonal antibody (Santa Cruz) at a dilution of 1:50, anti-SAP145 rabbit polyclonal antibody (Dr. R. Reed, Harvard Medical School), anti-hSm (Dr. P. Bubulya, WSU), anti-Sc35 mouse monoclonal antibody (Dr. P. Bubulya, WSU), and anti-Dnmt1 rabbit polyclonal antibody (Dr. W. Nelson, Johns Hopkins) at dilutions of 1:100.

Anti-Mcm3 rabbit polyclonal antibody, raised and purified as described previously (Ritzi et al.,

2003), was used at a dilution of 1:50. The secondary antibodies were FITC-conjugated donkey anti-mouse, Texas Red-conjugated donkey anti-mouse, FITC-conjugated donkey anti-rabbit,

Texas Red-conjugated donkey anti-rabbit, FITC-conjugated donkey anti-rat and Texas Red- conjugated donkey anti-human. All these secondary antibodies were obtained from Jackson

9 Immuno Research Laboratories Inc. or from Dr. P. Bubulya (WSU) and were used at dilutions of

1:200.

Images were obtained by using the Delta Vision Real Time Imaging System (Applied

Precision/Dr. P. Bubulya, WSU) with the Softworx Explorer Suite Software and Cool Snap camera kit included. A Plan-APO 60× oil / 1.4 numerical aperture objective lens was used.

DUE-B Purification Assay

HeLa, A1 and ∆CT cell lines were trypsynized, washed once with cold PBS, and then lysed with a lysis buffer containing 300 mM NaCl, 50 mM NaH 2PO 4 pH 8, 20 mM imidazole and 1%

NP40. Protease inhibitor cocktail (Sigma), 10 µl per 1 ml lysate, and 0.5 mM DTT were added to the lysis buffer. 1 ml lysis buffer was added for every 15 million cells. Cells were lysed for 1 hour rotating at 4˚C then centrifuged in an Eppendorf microcentrifuge at a maximum speed for 30 minutes at 4˚C. The supernatant was then incubated with the nickel nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) in a volume of 10 µl beads for ~1.5 ml whole cell extract for 2 hours at

4˚C to purify the 6xHis-tagged DUE-B protein. Following three washes with the lysis buffer containing 20 mM imidazole, 1X SDS sample buffer was added and the beads were boiled off for

3 minutes at 95˚C then centrifuged by an Eppendorf microcentrifuge at 3000 rpm for 2 minutes at

4˚C. The supernatant was then transferred to a microcentrifuge tube to be loaded on a gel.

Western blot Analysis

Purified samples were resolved on a 10% acrylamide SDS-PAGE gel for 1 hour at 115 volts and 2 hours at 180 volts. Proteins were then transferred to a PVDF membrane overnight at a constant current of 500 mA. Rabbit polyclonal anti-DUE-B antibody and anti-Orc2 antibody, raised and purified as described previously (Ritzi et al., 2003), were used at dilutions of 1:2000 in

5% nonfat dry milk in Tris buffered saline with tween 20 (TBST). Anti-SAP145 and anti-Dnmt1 rabbit polyclonal antibodies were both used at dilutions of 1:1000 in 5% nonfat dry milk in TBST.

10 The horseradish peroxidase-conjugated secondary antibodies were used at dilutions of 1:10,000 in

5% nonfat dry milk in TBST as well.

Mass Spectrometry Analysis Sample Preparation

HeLa cell lysate (2 mg), lysed as mentioned above, was incubated with 10 µl Ni-NTA beads for 2 hours then centrifuged by an Eppendorf microcentrifuge at 3000 rpm for 2 minutes at 4˚C.

The supernatant was then divided in two microcentrifuge tubes containing fresh 5 µl Ni-NTA beads each. 1 µg purified His-tagged Sf9 DUE-B, expressed from a baculovirus vector in insect

(Sf9) cells, was added to one of the two tubes, then the samples were incubated for 2 hours rotating at 4˚C. Beads were pelleted, washed 3 times with 20 mM imidazole then eluted with 250 mM imidazole (Figure 5). The eluted samples were characterized by mass spectrometry at The

Keck Foundation Biotechnology Resource Laboratory, Yale University.

HeLa Whole Cell Extract

Pre-clear with Ni-NTA beads

+Sf9 DUE-B - Sf9 DUE-B

+Ni-NTA beads 2 hrs

3X Washes with 20 mM Im

Elute with 250 mM Im

Mass Spectrometry

Figure 5: Mass spectrometry sample preparation procedure .

11 Cell Fractionation

To compare the proportion of DUE-B binding to chromatin and localized in speckles in A1 cells to that of ∆CT DUE-B in ∆CT cells, the cell fractionation method used to examine chromatin bound proteins (Mendez and Stillman, 2000) was implemented with some modifications. A1 and ∆CT cells, each grown in one 15 cm culture plate, were trypsinized then washed with cold PBS containing protease inhibitors (5 µg/ml Pepstatin A, 5 µg/ml Leupeptin, 5

µg/ml Aprotonin and 0.1 mM phenylmethylsulphonylfluoride [PMSF]). The cell suspension was centrifuged at 1300g for 4 minutes at 4˚C and resuspended in buffer A (10 mM HEPES (pH 8),

10 mM KCl, 1.5 mM MgCl 2, 0.34 M sucrose, 10% glycerol, 1 mM DTT and protease inhibitors).

0.1% Triton X-100 was added to the suspension and kept on ice for 10 minutes with several tube inversions. After centrifugation (1300g, 4 minutes at 4˚C), the pellet (P1) was resuspended in buffer A and divided in two microcentrifuge tubes. 300 units of RNase free DNase I (Takara) were added to one of the P1 suspension tubes with a buffer containing 100 mM Tris-HCl (pH7.5),

30 mM MgCl 2, 5 mM DTT and 15 mM CaCl 2, incubated for 10 minutes at 37˚C, 40 minutes at

4˚C then 10 minutes at 37˚C followed by centrifugation at 1300g for 4 minutes at 4˚C. The supernatant (S*) was preserved at -80˚C. The pellet (P*) was incubated in buffer B (15 mM Tris-

HCl (pH 7.5), 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT and protease inhibitors) for 30 minutes on ice, centrifuged at 1700g for 4 minutes at 4˚C then washed with buffer B yielding the pellet

(P3) and the supernatant (S3), which was kept at -80˚C. The pellet (P3) was further sonicated in

3X SDS-PAGE sample buffer at 40% altitude in an Ultrasonic Dismembranator (Fisher Scientific,

Model 500) for 5 minutes, 30 seconds on and 5 seconds off. The other P1 suspension tube (not being treated with DNase I) was centrifuged to give the supernatant (S**) to be frozen at -80˚C and the pellet (P**) to be processed in the same manner as the other P* explained earlier (see

Figure 18).

12 Gel Filtration Assay

A1 cells, trypsinized and washed with cold PBS, were lysed with Tissue Protein Extraction

Reagent (T-PER) (Pierce) by suspending 30 million cells in 1 ml T-PER buffer containing protease inhibitor cocktail 10 µl/ml and phosphatase inhibitor 10 µl/ml (Pierce) for 10 minutes on ice, then Dounce homogenizing (20 strokes) and centrifuging in an Eppendorf microcentrifuge at

3000 rpm for 5 minutes at 4˚ C. The supernatant (5 mg) was loaded on a Sepharose-200 column

(Amersham) packed in a buffer containing 150 mM NaCl, 10 mM Tris, pH 7.5 and 1 mM EDTA.

After collecting the fractions, a sample from every 3 rd fraction was taken to be analyzed by western blotting as described above.

13 Results

DUE-B is Localized in Nuclear Speckles in HeLa Cells

In order to understand DUE-B’s distribution in the cell better, and define its colocalized proteins, indirect immunofluorescence was performed by using rabbit polyclonal anti-DUE-B antibody (Figure 6A).

DUE-B DAPI merge A

α-DUE-B

α-His

Figure 6: DUE -B is in speckles in HeLa and A1 cells . A, HeLa cells fixed with 2% paraformaldehyde, permeabilized with 0.2 Triton X-100 then labeled with anti-DUE-B antibody. B, A1 cells (a HeLa cell line stably overexpressing a His-tagged DUE-B) were fixed then permeabilized as in “ A”, then labeled with anti-His antibody. C, same as “ B” but cells were pre- extracted with 0.2% Triton X-100 prior to fixation.

14 DUE-B appeared to be mainly nuclear, confirming what was shown previously in the biochemical experiments (Casper et al., 2005; Ghosh et al., 2006). When compared to DAPI staining, DUE-B was concentrated around the areas where DAPI was depleted, a typical characteristic of nuclear speckles (Hall et al., 2006). DUE-B’s observed pattern was distinct from other nuclear proteins such as the replication protein Mcm3, a member of the minichromosome maintenance complex involved in DNA replication (Figure 7). The nuclear speckled pattern, which was seen in HeLa cells incubated with anti-DUE-B antibody, was also observed by using mouse monoclonal anti-

His antibody on HeLa cells stably overexpressing His-tagged DUE-B (A1 cells). Since exogenously expressed His-tagged DUE-B efficiently dimerizes with endogenous DUE-B (Figure

15B), the anti-His antibody reveals the location of both His-tagged homodimers and heterodimers.

Mcm3 DAPI A

Figure 7: Mcm3 displays a B different distribution pattern than DUE-B. A, A1 cells fixed and permeabilized as in Figure 6, then labeled with anti-Mcm3 antibody. B, same as “ A” but cells were pre- extracted prior to fixation as in Figure 6.

To exclude the possibility that exogenous DUE-B was positioned in speckles due to its C- terminal 6xHistidine tag, HeLa cells were transiently transfected with pcDNA 3.1D/V5-His/ lac Z plasmid to be examined by the immunofluorescence microscopy. Using anti-His antibody, the

His-tagged LacZ transfected cells did not show the pattern observed for DUE-B (Figure 8A). The distinction between His-tagged DUE-B and the LacZ gene product, β-galactosidase, was yet more

15 evident when cells were pre-extracted with Triton-X100, where the signal corresponding to β- galactosidase was not visible (Figure 8B) while DUE-B’s speckles clearly stood out due to pre- extracting the nucleoplasmic fraction of DUE-B (Figure 6C). Other His-tagged proteins, known not to be in speckles, have also been detected by immunofluorescence and appeared in different distribution patterns (Marion et al., 1999). HeLa cells did not react with the anti-His antibody when the antibody was applied on the untransfected cells (Figure 8C) further confirming that the antibody was directed specifically to His-tagged DUE-B.

α-His DAPI merge A

Figure 8: Speckled pattern with His -antibody is speci fic to DUE -B. A, HeLa cells were transiently transfected with pcDNA 3.1D/V5-His/ lac Z plasmid, fixed then permeabilized as in Figure 6, then labeled with anti-His antibody. B, same as “A” but cells were pre- extracted prior to fixation as in Figure 6. C, HeLa cells, not pre-extacted, were labeled with anti-His antibody at a dilution of 1:200.

To obtain further evidence that DUE-B is found in IGCs, A1 cells were co-labeled with antibodies against both DUE-B and human Sm, a constituent of the small nuclear

16 ribonucleoproteins and known to be in speckles and in Cajal bodies (Nyman et al., 1986; Hackl, et al., 1994). The results showed that the two labeled proteins colocalized in nuclear speckles, whereas hSm appeared in Cajal bodies without the association of DUE-B signal (Figure 9).

DUE-B hSm merge

Figure 9: WT DUE -B colocalizaton in speckles with hSm . A1 cells were pre-extracted, fixed then permeabilized as in Figure 6, then they were co-labeled with anti-His and human anti-Sm antibodies. Arrows show Cajal bodies where only hSm is seen.

The previous immunofluorescence results together suggest that a considerable fraction of nuclear

DUE-B is localized in the interchromatin granule clusters, while another part is loosely bound to chromatin or in the nucleoplasm.

SAP145 Co-Purifies with His-tagged DUE-B

Earlier data showed that a small portion of DUE-B was found to elute in a high molecular weight fraction by gel filtration (Casper et al., 2005) indicating the possibility of DUE-B’s interaction with other proteins. As an attempt to recognize any probable interacting proteins, Sf9

DUE-B, was added to HeLa whole cell extract, incubated with Ni-NTA beads then eluted from the beads and sent for mass spectrometry analysis. LCMS/MS analysis (Keck Facility, MA) was performed on DUE-B plus HeLa extract elution and the elution from HeLa extract control. The

17 search results revealed the presence of 2 peptides pertaining to the splicing factor 3B subunit 2

(SAP145) in the DUE-B plus HeLa extract but not in the control (Figure 10).

Figure 10: Mass spectrometry results : SAP145 is co-purified with DUE-B by the Ni-NTA beads pull down assay.

Subsequently, when the primary gel filtration experiment was repeated, a proportion of SAP145 was found in the same high molecular weight fraction as that of DUE-B (Figure 11). These results support the mass spectrometry findings, since SAP145 co-eluted with the His-tagged

DUE-B in both experiments.

18 Aldolase Catalase (150kD) (232kD) Figure 11: WT DUE -B co -elutes with SAP145. A1 whole cell extract (5 mg), lysed with T-PER buffer, was loaded on a SAP145 Sephacryl 200 chromatography column. Samples from every third fraction were loaded on a gel then blotted for DUE-B and SAP145. DUE-B

DUE-B Colocalizes with SAP145 in Speckles

Based on the mass spectrometry results and the presence of SAP145 mainly in nuclear speckles (Schmidt-Zachmann et al., 1998), A1 cells were co-labeled with anti-SAP145 and anti-

His antibodies and examined by immunofluorescence microscopy. As seen in Figure (12), results showed that both proteins clearly localized in the IGCs.

DUE-B SAP145 merge

Figure 12: WT DUE -B colocalizes with SAP145. A1 cells were pre-extracted, fixed then permeabilized as in Figure 6, then they were co-labeled with anti-His and anti-SAP145 antibodies.

C-Terminus Deleted DUE-B Mutant Is More Concentrated in Nuclear Speckles than the

Wild-Type

Since human DUE-B has ~ 60 extra C-terminal amino acids when compared to its yeast orthologue (Casper et al., 2005), a DUE-B mutant lacking this C-terminus ( ∆CT) was constructed

19 (Figure 2) (Kemp et al., 2007) in order to study the effects of this deletion on DUE-B’s suggested function in replication, especially since DUE-B is thought to bind to DNA via this carboxy- terminus (Kemp et al., 2007). Knowing that DUE-B’s ortholgue in yeast (DTD1) was shown to be cytoplasmic (Huh et al., 2003), we sought to examine whether the extra 62 residues in hDUE-

B were responsible for its presence in the nucleus. To do so, immunofluorescence on HeLa cells stably expressing the His-tagged mutant ∆CT DUE-B was performed by using anti-His antibody.

Surprisingly, ∆CT DUE-B was almost confined to the nuclear speckles (Figure 13). This observation was confirmed by its colocalization with hSm (Figure l4A) and SAP145 (Figure 14B).

∆CT DUE-B DAPI merge A

Figure 13: CT DUE -B in nuclear speckles . A, CT cells (a HeLa cell line stably expressing DUE-B lacking the last 62 carboxy terminal amino acids) were fixed, permeabilized as in Figure 6, then they were labeled with anti-His antibody at a dilution of 1:10. B, image in “ A” in the deconvolved form.

20 A ∆CT DUE -B hSm merge

B ∆CT DUE -B SAP145 DAPI merge

Figure 14: DUE-B colocalization in speckles with SAP145 and hSm. A, ∆CT cells were fixed then permeabilized as in Figure 6, then they were co-labeled with anti-His and anti-Sm antibodies at dilutions of 1:10 and 1:100 respectively. B, ∆ CT cell were pre-extracted prior to fixation as in Figure 6, then they were co-labeled with anti-His antibody at a dilution of 1:200 and anti-SAP145 antibody at a dilution of 1:100. White arrows indicate colocalization, blue arrow heads show the presence of hSm (A) or SAP145 (B) alone, and long blue arrows show DUE-B’s presence alone.

As mentioned earlier, A1 DUE-B was observed in both nuclear speckles and diffused in the nucleoplasm, whereas ∆CT DUE-B is concentrated in the IGCs (Figure 15A). A1 cells needed to be pre-extracted with Triton X-100 to obtain the clear speckled image (Figure 15A panel c).

Given that DUE-B is a homodimer and that ∆CT DUE-B’s expression is approximately equal to or less than that of the endogenous (Figure 15B, M. Kemp, unpublished data), then the His- antibody signal in speckles corresponds equally to ∆CT DUE-B homodimer as well as the dimer of ∆CT and endogenous DUE-B molecules. In conclusion, since ∆CT DUE-B mutant is still nuclear despite lacking its carboxy terminus while its yeast orthologue is cytoplasmic, the

21 carboxy terminus might not be the only factor necessary for recruiting DUE-B to the nucleus and speckles.

A WT DUE -B ∆CT DUE -B a b

His-tagged tagged EndogenousDUE-B DUE-B c d

ΔCT DUE-B

WT ΔCT

Figure 15: ∆∆∆CT DUE-B is more enriched in speckles than the WT DUE-B. A, A1 (a and c) and ∆CT cells ( b and d) were fixed then permeabilized as in Figure 6, then they were labeled with anti-His antibody ( a, c and d at a dilution of 1:200 and b at a dilution of 1:10. In c and d, cells were pre-extracted prior to fixation as in Figure 6.

DUE-B Does not Appear in Replication Sites by Immunofluorescence or Change Its

Speckled Pattern at Late G1/Early S

Based on DUE-B’s proposed role in replication (Casper et al., 2005) and its ability to bind

DNA (Ghosh et al., 2006), we sought to look for DUE-B at replication sites and see whether the mutant ∆CT would appear differently. Bromodeoxyuridine incorporation followed by indirect immunofluorescence showed that DUE-B, in both A1 and ∆CT cells, was not visible at the BrdU labeled sites (Figure 16A, B).

A1 cells were also synchronized at late G1 and early S phases to study DUE-B’s distribution by immunofluorescence at the time of replication initiation. This was of a particular interest since

DUE-B was found to be enriched in the c-myc replication origin as well as at the lamin B2 origin

22 (Casper et al., 2005; Ghosh et al., 2006). DUE-B’s localization in speckles was similar in G1 and

S phase cells (Figure 16C, D).

α-BrdU α-His merge A WT

B ∆CT

DUE-B/DAPI

C Figure 16: DUE -B patterns in different cell cycle phases. A and B, DUE-B is not visible in replication sites. A1 and ∆CT cells were incubated with 10 µm bromodeoxyuridine for 20 minutes prior to pre-extraction, fixation and permeabilization (as in Figure 6). Cells were then incubated with anti-BrdU,anti-His antibodies and 10 units DNase I for 2 hours. C and D, DUE-B’s speckled pattern does not change in cells arrested in G1/early S. A1 D cells were treated with 1µg/ml aphidicolin (C) or 0.2 M mimosine (D) for 18 hours prior to pre-extraction. After fixation and permeabilization (as in Figure 6), cells were labeled with anti-His antibody. Synchronizations by aphidicolin ( C) and mimosine ( D) were confirmed by folw cytometry.

23 In mitotic cells however, DUE-B became diffusely distributed in the mitotic cytoplasm (Figure

17A, B) resembling in this situation other speckle components which disperse as small granules in the cytoplasm after chromatin condensation and nuclear membrane breakdown (Bubulya et al.,

2004; Thiry, 1995; Ferriera et al., 1994).

DUE -B DUE -B/DAPI SAP145/DAPI A

Figure 17: DUE -B loses concentration in speckles during mitosis. A1 cells were pre-extracted, fixed then permeabilized as in Figure 6. Cells were then co-labeled with anti-His and SAP145 antibodies. Arrows show A, a cell in telophase and B, cells exiting mitosis.

DUE-B in the Nucleus Is on DNA and in Nuclear Speckles

Previously, treatment of HeLa nuclei with micrococcal nuclease (MNase) showed that DUE-

B became soluble upon DNA digestion (Casper et al., 2005), thus, strongly suggesting that DUE-

B is bound to DNA. The immunofluorescence findings, however, suggest that DUE-B in the

24 nucleus is distributed between the nucleoplasm and the nuclear speckles. Upon cellular fractionation, the chromatin fraction contains other nuclear components including IGCs. This is because the chromatin fraction is obtained by lysing nuclei then pelleting the nuclear lysate

(Mendez and Stillman, 2000), whereas IGCs’ isolation requires further extraction after nuclei treatment with nucleases (Mintz et al., 1999) (Figure 18).

Cell fractionation procedure IGCs Purification Procedure Lyse cells with non-ionic detergent 0.1% Triton X100 10 min/ice

Centrifuge 4 min/4C/1300g

S1 P1

+DNase I 10 u/µl /1hr/4 ˚C

+DNase I -DNase I 0.5 M NaCl 10 min at 37 ˚C, 3X5 min/ice 30 min at 4 ˚C then 10 min at 4 ˚C

Centrifuge Centrifuge 4 min/4C/1300g 4 min/4C/1300g

S* P* S** P**

0.25 M Cs 2SO 2 Resuspend in EDTA/EGTA Resuspend in EDTA/EGTA Mechanical 30 min/ice 30 min/ice disruption Centrifuge 20,800g/2 min

S3 P3 S3 P3 S P IGCs Nuclear lamina,nuclei and other nuclearcomponents

Figure 18: Cell fractionation procedure. A comparison between the cell fractionation procedure performed here and the IGCs purification procedure as described in Mintz et al, 1999.

Such an understanding was supported by finding SAP145 in the chromatin fraction (Figure 19A), a result that suggested that DUE-B present in this fraction could be bound to both chromatin and nuclear speckles. To gain a better understanding of DUE-B’s distribution in the chromatin fraction between speckles and DNA, A1 and ∆CT fractions were compared with and without

25 DNase I treatment to see if more ∆CT DUE-B would remain in the chromatin fraction despite

DNA digestion. RNase free DNase I was used instead of MNase to resemble the DNA digestion

used in the IGCs extraction. Results showed no difference between both DUE-B types (Figure

19B, C). They were both very sensitive to DNase I treatment. A considerable amount of Orc2 was

not released by DNase I digestion suggesting its association to nuclease-resistant structures. This

finding has been also observed in other studies (Fujita et al., 2002; Mendez and Stillman, 2000).

A Figure 19: DUE -B in chromatin fraction is sensitive to DNase I α -SAP145 treatment and contains speckled proteins. A, samples previously prepared (M. Kemp) were loaded on a 10% polyacrylamide gel then probed for DUE-B and SAP145. B, A1 cells (10 7) were fractionated according to the cell fractionation procedure presented in figure 6, loaded on a 10% polyacrylamide gel then incubated with anti-DUE-B α -DUE-B and anti-Orc2 antibodies C, same as “B ” but ∆CT cells were used instead of A1 cells. P3 S2 WCE

B C Orc2 Orc2

His-tagged DUE -B Endogenous Endogenous DUE-B DUE-B

ΔCT DUE-B P3 S3 S* P3 S3 S** P3 S3 S* P3 S3 S**

DNase I + + + - - - DNase I + + + - - -

The apparent sensitivity of DUE-B to DNase I treatment indicates that this “chromatin fraction”

represents mainly the DNA-bound DUE-B; alternatively, DUE-B may be a nuclease-sensitive

component of IGCs or it may be released by Triton X-100 from nuclear speckles due to its

association with low affinity in the IGCs, as has been the case for other proteins found to be in

speckles but not detected in the IGCs chemically (Saitoh, 2004).

DUE-B Colocalizes Partially with Dnmt1 in Speckles

Earlier experiments using mass spectrometry after Ni-NTA pull down from A1 whole cell extract (Kemp, unpublished) or immunoprecipitation from Xenopous egg extract showed that

DUE-B interacts with Dnmt1 (Figure 20A: Kemp and Leffak, unpublished data). The results were also confirmed by Ni-NTA pull down of Sf9 DUE-B added to HeLa whole cell extract (Figure

20B).

A B Ni-NTA pull down hDnmt1

WCE HeLa Clone A1 α -Dnmt1

Ni-NTA Pull-down HeLa WCE WCE +Sf9 -Sf9 DUE-B DUE-B

Figure 20: DUE -B interacts with Dnmt1. B, 4 mg of HeLa whole xDnmt1 cell extract, lysed as in Figure 16, was incubated with Ni-NTA for 2 hours after adding 2 μg Sf9 DUE- HSS NRS Anti-DUE-B B to one of the two HeLa extract tubes. Eluates were resolved on a Immunoprecipitate 10% polyacrylamide gel and probed for hDnmt1 by using M. Kemp, Ph.D. anti-Dnmt1antibody.

Since DUE-B’s nuclear distribution pattern appeared to be both diffuse and in speckles, immunofluorescence microscopy was used to study where its interaction with Dnmt1 is located.

In previous studies, Dnmt1 examined in mouse fibroblasts was known to have a diffuse pattern in the nucleus. During S phase, it localized to distinct foci around some replication sites, presumably, in order to maintain DNA methylation once replication occurs (Bestor, 2000; Leonhardt et al.,

27 1992). In the experiments performed on A1 cells, Dnmt1 also appeared to colocalize to some replication foci (Figure 21).

Dnmt1 BrdU merge DAPI

Figure 21: Dnmt1 is seen at some replication foci in A1 cells . A1 cells were treated with BrdU, pre-extracted, fixed then permeabilized as in Figure 16. Then cells were co-labeled with anti-Dnmt1, anti-BrdU and Dnase I as in Figure 16.

To examine DUE-B and Dnmt1 colocalization, A1 and ∆CT cells were co-labeled with anti-His and anti-Dnmt1 antibodies. Dnmt1 colocalized partially with both A1 DUE-B and ∆CT DUE-B in many cells (Figure 22). It appeared that the two proteins interacted in the nuclear speckles, a result rather surprising because Dnmt1 is not known to be in speckles.

DUE-B Dnmt1 merge A WT

B ∆CT

Figure 22: DUE -B partially colocalizes with Dnmt1. A, A1 cells were pre-extracted, fixed, permeabilized as in Figure 6. Cells were then co-labeled with anti-His and anti-Dnmt1 antibodies. White arrows indicate colocalization, blue arrows show DUE-B’s presence alone and blue arrow heads show the localization of Dnmt1 alone.

To confirm this observation, A1 cells were co-labeled with SC35 and Dnmt1 antibodies. Dnmt1 clearly colocalized with SC35 in some cells (Figure 23). Dnmt1 also showed the same speckled pattern in a percentage of HeLa cells where no exogenous DUE-B is over-expressed (Figure 24).

In all immunofluorescence experiments performed, Dnmt1 appeared to be in speckles in a certain percentage of the cells examined, possibly at a particular phase of the cell cycle.

Dnmt1 SC35 merge DAPI

Figure 23: Dnmt1 appears in speckles in some cells. A1 cells were pre-extracted, fixed then permeabilized as in Figure 6. Then they were co-labeled with anti-Dnmt1 and anti-SC35.

30 Dnmt1 DAPI A

Figure 24: Dnmt1 appears appears Hela in speckles in HeLa cells, A1 and ∆∆∆CT cells labeled only with anti-Dnmt1 antibody. A, HeLa cells were pre-extracted, fixed then permeabilized as in B Figure 6, then they were labeled with anti-Dnmt1 antibody alone. B, and A1 C, same as in “ A”, but cells used were A1 and ∆ CT cells respectively.

∆CT

DUE-B Does not Colocalize with PML-NB

As mentioned earlier, DUE-B interacted with Dnmt1; however, it was not visualized at replication foci, the anticipated interaction site for DUE-B and Dnmt1. On the other hand, Dnmt1, previously found at replication sites, has been recently observed at sites of DNA repair

(Mortusewicz et al., 2005). Some evidence also predicted a correlation between Dnmt1 and promyelocytic leukemia (PML) protein (Di Croce, 2002), the main constituent of PML-NBs

(Dellaire et al., 2004). For the above reasons, an immunofluorescence experiment was performed on A1 cells to test whether DUE-B colocalizes with PML. Results, however, did not reveal any

31 colocalization (Figure 25A). The experiment was repeated after applying UV-irradiation. There was no significant change in DUE-B’s distribution pattern observed after DNA damage induction by UV (Figure 25B).

A DUE -B PML merge DAPI

B DUE -B/PML

a b

Figure 25: A, DUE -B does not colocalize with PML -NB. A1 cells, pre-extracted, fixed then permeabilized as in Figure 6, were co-labeled with anti-His antibody and anti-PML antibody. “B”, UV-Treatment does not change DUE-B’s distribution pattern. Immunofluorescence experiment was performed as in “ A”. In “ b”, cells were treated with 60 J/m² 2 hours before the experiment.

32 Discussion

By using two different antibodies against DUE-B, immunofluorescence revealed that DUE-B resides in nuclear speckles and colocalizes with two known splicing proteins: hSm and SAP145.

Such a finding was quite interesting since DUE-B is suggested to have a role in replication while speckles mainly contain the splicing factors involved in mRNA processing. Nevertheless, the study performed by Saitoh et al. (2004) identified proteins involved in other nuclear functions as members of the IGCs. Several DNA repair proteins were suggested to be in nuclear speckles such as XPE UV-damaged DNA binding protein and XPA binding protein 2. As mentioned earlier, not detecting DUE-B in that study might have been due to its association with IGCs with low affinity; hence, it might dissociate during purification. This explanation is consistent with the fractionation experiment results discussed below. Furthermore, DTD1 DUE-B’s orthologue in yeast was found to interact with RBP9, a subunit of RNA polymerase II that has been reported to be part of the

IGCs in several studies (Mortillaro et al., 1996; Bregman et al., 1995: Saitoh et al., 2004).

Although RNA polymerase II was not observed to interact or co-purify with DUE-B the above data supports the presence of DUE-B in speckles.

The speckled DUE-B distribution observed by indirect immunofluorescence was supported by the mass spectrometry results, which showed that SAP145 co-purified with the His-tagged Sf9

DUE-B. The study of Tran et al. (2004) showed that SAP145 co-purified with protein phosphatases complex including protein phosphatase 2A (PP2A). Interestingly, the DUE-B orthologue in yeast, DTD1, was shown to interact with a protein phosphatase 2A named Sit4

(Gavin et al., 2002), which has a proposed roles in DNA repair (Douvolle, 2004). The C-terminal amino acid sequence of hDUE-B also possesses a significant similarity to the PP2A target sequence. Being conscious of both studies, the PP2A target sequence in DUE-B and the mass spectrometry finding of SAP145’s co-purification with DUE-B together suggest that DUE-B might be part of the PP2A/SAPs complex. Such an explanation is supported by the gel filtration

33 result of DUE-B’s elution in the high molecular weight fraction, especially that SAP145 was found in the same fraction of DUE-B when the experiment was repeated.

DUE-B was previously suggested to interact with Dnmt1 (Kemp and Leffak, unpublished data). Immunofluorescence showed that this interaction is most likely occurring in speckles.

Dnmt1, as suggested by Bestor (2000), is known to have two different nuclear distributions according to the cell cycle. Generally, Dnmt1 has a diffuse nuclear pattern. During S phase

Dnmt1 appeared to form foci around γ-satellite DNA in mouse fibroblasts (Bestor, 2000). In the immunofluorescence experiments performed in this study, Dnmt1 appeared in both partial replication foci localization pattern and the nuclear diffuse pattern in HeLa cells. Additionally, a percentage of the cells examined had Dnmt1 in nuclear speckles. Further experiments need to be performed to confirm Dnmt1 presence in speckles and recognize its function there. Nevertheless, such an observation could be explained in light of relevant studies.

Dnmt1 was recruited to DNA repair sites (Mortusewicz et al., 2005). Moreover, upon DNA damage, Dnmt1 was recently shown to down regulate Cdc25 and Cdc2, two important factors that mediate entry into mitosis (Le Gac et al., 2006). Notably, Cdc2 has been observed in nuclear speckles upon UVB-induced G2 arrest (Fayolle et al., 2006). SAP145 also appears to have a role in cell cycle progression. A study performed by Terada et al. (2006) showed that the depletion of

SAP145 activated a checkpoint-mediated cell cycle arrest through the induction of BRCA1 and gamma H2AX. One of the consequences of BRCA1-mediated G2/M cell cycle arrest is the down- regulation of Cdc25 (Yan et al., 2005). Considering all the above, the presence of Dnmt1 in nuclear speckles might be due to the speckles’ proposed role in DNA damage response, possibly via SAP145. It is worth testing whether DUE-B, co-localized with SAP145 and Dnmt1 in speckles, is co-functioning with these proteins at G2 checkpoint activation. This supports the suggested correlation between the different nuclear functions such as the one predicted between transcription, repair and apoptosis, which was based on the presence of the SAP motif identified in the proteins as a DNA binding motif (Aravind and Koonin 2000). Interestingly, both Dnmt1

34 and DUE-B possess a motif with some similarity to the SAP motif as found in SAP145. It has also been observed that proteins containing the SAP motif frequently possess another motif that is related to DNA processing. Thus, these proteins are suggested to have in common the ability to associate with both DNA and RNA (Saitoh et al., 2004). Likewise, DUE-B can bind tRNA as well as DNA, as evidenced by its tRNA deacylase and origin binding activities (Kemp et al.

2007). DUE-B therefore shares another feature with the SAP motif-possessing speckle proteins.

An alternative explanation for the detection of Dnmt1 in speckles can be due to its potential role in transcription repression where nuclear speckles would serve as a storage site for Dnmt1 like other transcription factors found there (Saitoh et al., 2004). Along with Dnmt3b, Dnmt1 was found in HeLa cell nucleoli and suggested to be involved in ribosomal transcription regulation

(Majumder et al., 2006). Szyf and collegues also suggested a role for Dnmt1 in transcription repression by a mechanism independent of DNA methylation (Milutinivic et al., 2004).

Additionally, the expression of the PML-RAR oncogene has been shown to alter the nuclear compartmentalization of Dnmt’s and recruit them to the newly formed PML-RAR microspeckles

(Di Croce et al., 2002). The PML-RAR repressed transcription and blocked differentiation through the recruitment of Dnmt’s and subsequent promoter methylation. It would be interesting to test whether DUE-B might be involved in such a pathway since it seems to interact with

Dnmt1. Inducing DNA damage in HeLa cells by UV neither changed DUE-B’s distribution in the nucleus, nor that of PML. Such an experiment could be re-performed in a p53+ cell line since

PML response to UV induced damage is p53 dependent (Seker et al., 2003) and p53 levels are low in HeLa cells due to the presence of the HPV E6 protein (Scheffner at al., 1991).

∆CT DUE-B was seen almost exclusively in speckles by immunofluorescence, whereas the full-length form was observed both in speckles and diffuse in the nucleus. As mentioned earlier,

DUE-B was shown to be phosphorylated. Its C-terminus is suggested to possess phosphorylated residues and appears to have the consensus sequences for both casine kinase II and PP2A activities (Kemp, unpublished data). Therefore, it is possible that DUE-B becomes activated and

35 moves to its functioning site by either phosphorylation or dephosphorylation, hence, resembling other IGCs’ proteins whose function and localization is directed by phosphorylation (Hall et al.,

2006; Bubulya et al., 2004; Dirks et al., 1999). Losing the C-terminus might render DUE-B insensitive to phosphorylation; thus, it stays in speckles.

DUE-B in the chromatin fraction was sensitive to DNase I treatment which strongly supports its binding to DNA. On the other hand, DUE-B was not detected in replication foci by immunofluorescence in spite of the primary chemical experiments that strongly suggest its role in replication initiation regulation. However, other replication initiation proteins such as Mcm3 did not colocalize with replication foci either (Krude, et al., 1996; Dimitrova and Berezney, 2002).

Such a resemblance to Mcm3 is consistent with the findings that showed DUE-B to bind to c-myc origin in a pattern similar to Mcm3 (Ghosh et al., 2006). Also, previous experiments showed that

DUE-B binds DNA between Mcm loading (pre-RC formation) and RPA loading (DNA unwinding) (Casper et al., 2005; Ghosh et al., 2006), consistent with the binding of DUE-B to double stranded DNA (Kemp et al., 2007). The lack of localization of DUE-B at active replication foci may be explained by the release of DUE-B upon DNA unwinding, prior to initiation of nucleotide incorporation. Not detecting DUE-B in replication foci, however, still does not rule out its presence there, especially that it was found to interact with Dnmt1 whose observation in replication foci has been well recognized as stated earlier. It is possible that DUE-

B’s 6xHis-tag might have been masked by DNA binding, especially that the C-terminus is suggested as DUE-B’s DNA binding region. Besides, anti-DUE-B antibody was not very helpful in the immunofluorescence experiments. Therefore, affinity purification of this antibody might render it more specific and, thus, enable the visualization of DUE-B in its proposed functional sites. ∆CT DUE-B in chromatin fraction was also sensitive to DNase I treatment despite the suggestion that its carboxy terminus is needed for DNA binding. This result is consistent with a

DUE-B siRNA experiment (Kemp and Leffak, unpublished data), which showed that ∆CT DUE-

36 B is still on DNA despite the endogenous DUE-B knock down. Thus, other factors might be important for DNA binding along with the C-terminus such as the SAP motif discussed above.

In summary, immunofluorescence showed that DUE-B is localized predominantly in speckles, with SAP145 and partially with Dnmt1. It was not visualized in replication foci; however, cellular fractionation strongly supports its binding to DNA. Considering the related literature, these results could indicate a possible role for DUE-B in DNA repair or in the initiation step of DNA replication. Further studies might reveal whether DUE-B’s existence in speckles is due to its co- involvement in DNA repair with SAP145 and/or Dnmt1.

37 References

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