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(The FASEB Journal. 2007;21:26-34.)
© 2007 FASEB

Methylation of DNA polymerase ß by protein arginine methyltransferase 1 regulates its binding to proliferating cell nuclear antigen

Nazim El-Andaloussi*,1, Taras Valovka*,1, Magali Toueille*, Paul O. Hassa*, Peter Gehrig{dagger}, Marcela Covic*, Ulrich Hübscher* and Michael O. Hottiger*,2

* Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich, Switzerland; and

{dagger} Functional Genomics Center Zurich, Zurich, Switzerland

2Correspondence: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Email: hottiger{at}vetbio.unizh.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA polymerase ß (pol ß) is a key player in DNA base excision repair (BER). Here, we describe the complex formation of pol ß with the protein arginine methyltransferase 1 (PRMT1). PRMT1 specifically methylated pol ß in vitro and in vivo. Arginine 137 was identified in pol ß as an important target for methylation by PRMT1. Neither the polymerase nor the dRP-lyase activities of pol ß were affected by PRMT1 methylation. However, this modification abolished the interaction of pol ß with proliferating cell nuclear antigen (PCNA). Together, our results provide evidence that PRMT1 methylation of pol ß might play a regulatory role in BER by preventing the involvement of pol ß in PCNA-dependent DNA metabolic events.—El-Andaloussi, N., Valovka, T., Toueille, M., Hassa, P. O., Gehrig, P., Covic, M., Hübscher, U., Hottiger, M. O. Methylation of DNA polymerase ß by protein arginine methyltransferase 1 regulates its binding to proliferating cell nuclear antigen.


Key Words: posttranslational modification • DNA repair • protein interactions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAMMALIAN CELLS HAVE DEVELOPED multiple mechanisms for the maintenance of integrity of their genome (1) . The repair of single-base lesions and DNA single strand breaks is accomplished through DNA base excision repair (BER) (2) . BER is initiated by a DNA glycosylase that cleaves the N-glycosydic bond of a damaged base, producing an apurinic/apyrimidinic site, also known as an abasic site or activating protein (AP) site (3 , 4) . Subsequently, AP endonuclease 1 (APE1) cleaves the sugar-phosphate backbone of the DNA at the 5'-side of the AP site resulting in 3'-hydroxyl and 5'-deoxyribose phosphate (5'dRP) groups at the margins of a one-nucleotide gap; refs 5 , 6 ). At this point, the repair will proceed through at least two subpathways, designated as "short patch" BER (SP-BER) and "long patch" BER (LP-BER) (1) . The two pathways are distinguished not only by the repair patch sizes but also by their contributing factors (7 8 9) . In SP-BER, DNA polymerase ß (pol ß) inserts one nucleotide into the gap generating a nicked DNA intermediate with a 5'-dRP flap, which is subsequently removed by its associated dRP-lyase activity (10 , 11) , resulting in a ligatable nick that will be sealed by XRCC1/LigaseIII. It has been proposed that, in LP-BER, pol ß performs strand displacement synthesis generating a flap that is then removed by flap endonuclease 1 (Fen1; ref. 9 , 12 ). Thus, pol ß is considered as a key player in both SP-BER and LP-BER (13 14 15 16) . Consistent with this, mouse knockouts deficient for pol ß are nonviable and fibroblast cell lines derived from pol ß deficient embryos display marked hypersensitivity toward monofunctional DNA alkylating agents (14) . Depending on the involvement of proliferating cell nuclear antigen (PCNA), LP-BER has been further classified into a PCNA-dependent subpathway, where pol {epsilon}/{delta} are involved, and a PCNA-independent subpathway, where pol ß is the only DNA polymerase that mediates LP-BER DNA synthesis (12 , 17 18 19) . However, a recent study has demonstrated that PCNA can physically interact with pol ß (20) , raising the possibility that PCNA may have a direct or indirect role in pol ß-dependent BER.

Mammalian pol ß is a small constitutively expressed DNA polymerase (21) . It is a multifunctional enzyme represented by a single polypeptide consisting of 335 amino acids that build two functional domains connected by a protease sensitive hinge region (22) . The N-terminal 8 kDa domain harbors the dRP-lyase and the DNA binding activities, whereas the C-terminal 31 kDa domain constitutes the nucleotidyltransferase activity of the polymerase (11 , 23) . The resolution of the structure of pol ß has revealed that the 31 kDa domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the architecture of the Klenow fragment of Escherichia coli pol I (24) . The secondary structure of the fingers subdomain is represented by {alpha}-helixes, whereas palm and thumb subdomains consist of {alpha}-helixes and ß-sheets. These subdomains contain several structural elements important for ssDNA and dsDNA binding, binding of Mg2+ ions and dNTP selection. Additionally, pol ß contains three PCNA interacting motifs (PIMs) in {alpha}-helix I (fingers), {alpha}-helix L (palm), and {alpha}-helix M (thumb) that are designated I, II and III, respectively (20) .

Protein arginine methylation is a posttranslational modification that results in mono-methylated and symmetrical or asymmetrical dimethylated arginines (25) . In mammalians, protein arginine methyltransferases (PRMTs) represent a family of eight known enzymes that utilize S-adenosyl methionine as a methyl donor (26) . Among them PRMT1 is the predominant PRMT regarding measurable methyltransferase activity (27) . A considerable amount of PRMT1-dependent functions connects this enzyme to the process of gene regulation (28) . PRMT1 has also been implicated in a variety of other cellular processes, including signal transduction and DNA repair (29 , 30) . Originally, PRMT1 was identified in a yeast-two hybrid screen as an interacting partner of immediate-early gene and leukemia-associated gene products, TIS1 and BTG1, respectively (31) . Later studies have demonstrated that PRMT1 represents an arginine methyltransferase that specifically methylates arginine 3 in histone H4 in vitro and in vivo (32) . PRMT1 is also known to form a complex with the cytoplasmic domain of IFN receptor {alpha}/ß (33) . Down-regulation of PRMT1 activity by an antisense oligonucleotide resulted in a reduced sensitivity of the cells to the antiproliferative effect of IFN{alpha}/ß. Interleukin (IL) enhancer-binding factor 3 (ILF3) is another protein that interacts with and is specifically methylated by PRMT1. As a part of this functional interaction, ILF3 was shown to modulate PRMT1 activity in an in vitro methylation assay (34) . Transcription factor STAT1, Src kinase adapter protein Sam68, and scaffold attachment factor SAF-A were also found to interact with PRMT1 (35 36 37) . Recent findings indicate that PRMT1 methylates the DNA damage checkpoint factor Mre11 and regulates the exonuclease activity of this protein (38) . Cells containing hypomethylated Mre11 displayed intra-S-phase DNA damage checkpoint defects suggesting the involvement of PRMT1 in DNA damage responses (38) .

Several BER proteins, including pol ß, are regulated by posttranslational modifications (39) . Inactivation of pol ß activity by in vitro phosphorylation has previously been described (40) . Additionally, we provided an earlier evidence that pol ß is acetylated by the transcriptional coactivator p300 (41) . In vitro acetylation of K72 residue specifically reduced the dRP-lyase activity of pol ß, whereas its polymerase and AP-lyase activities were largely unaffected. Very recently, we have reported that pol ß formed a complex with PRMT6 and was methylated by PRMT6 (42) . PRMT6-mediated methylation specifically enhanced the polymerase activity of pol ß due to enhanced DNA binding and processivity. Mutation of methylation sites identified in pol ß significantly reduced the ability of the enzyme to protect complemented pol ß–/– cells against MMS-induced DNA damage.

In this study, we provide evidence that pol ß forms a specific complex with PRMT1 and is methylated by this methyltransferase. Mapping of the methylation site revealed that arginine 137 is an important target for PRMT1. We show that PRMT1-mediated methylation does not influence specific intrinsic activities of pol ß. However, the binding of pol ß to PCNA was severely affected by PRMT1-mediated methylation of arginine 137. Together, our results implicate that PRMT1 might play an important role in the regulation of pol ß/PCNA interaction and thus PCNA-dependent processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides and plasmids
The full-length pol ß and the pol ß {Delta} lyase domains were cloned into pQE30 vector (Invitrogen, Carlsbad, CA) as described previously (41) . Pol ß {Delta} Thumb and pol ß {Delta} Thumb{Delta} Palm fragments were amplified by polymerase chain reaction (PCR) from the pQE30–6HIS-pol ß plasmid and then were cloned into BamHI site of pQE30 vector. The pol ß R137K mutant was generated by site directed mutagenesis from the pQE30–6HIS-pol ß plasmid.

The 73mer and the corresponding 17mer primer substrates for DNA polymerase assays as well as the uracil containing 34mer oligonucleotide and its complementary strand harboring guanidine opposite to the uracil base were chemically synthesized and purified on denaturing polyacrylamide gels by Microsynth (Balgach, Switzerland). The sequences are the following: 73mer, 5'-GATCGGGAGGGTAGGAATATTGAGG ATGAAAGGGTTGAGTTGAGTGGAGATAGTGGA-GGGTAGTATGGTGGATA-3'; 17mer, 5'-TATCCACCATACTACCC-3'; and 34mer, 5'-CTGCAGCTGATGCGCUGTAC GGATCCCCGGGTAC-3'.

Proteins
N-terminal 6His-tagged full-length human pol ß and pol ß mutants were expressed in E. coli TG1 strain and purified by Fast Protein Liquid Chromatography on Ni-NTA HiTrap (Amersham Biosciences, Piscataway, NJ, USA) and Mono-SP HiTrap columns (Amersham Biosciences). N-terminal 6His-tagged Fen1 was overexpressed in E. coli BL21(DE3) pLysS strain and purified as mentioned above. Glutathione S-transferase (GST) fusion proteins were expressed in E. coli TG1 strain or SF9 insect cells and purified on Ni-NTA ProBond Resin (Invitrogen). Calf thymus histones (Type II-AS) were purchased from Sigma. Uracil-DNA glycosylase (UDG) from E. coli was purchased from Enzymax. Human APE1 was expressed in E. coli BL21(DE3) and purified as described previously (41) .

Antibodies
Pol ß specific antibody (Ab; 18S) was purchased from Neo Markers (Freemont, CA, USA). Mouse IgG (sc-2025), antitubulin (sc-8035), anti-PCNA, and antic-myc IgG (sc-9E10) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-hemagglutinin Ab was supplied by Covance.

Cells
The 293T cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX, 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

GST pull down and immunoprecipitation
GST-PRMT1 or GST alone bound to glutathione-sepharose beads were incubated with the indicated proteins in 50 mM HEPES (pH 8.0), 90 mM NaCl, 0.1% (v/v) Nonidet P-40, 1 mM DTT, 1 mM PMSF, protease inhibitors (2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml bestatin) for 2 h at 4°C. The beads were washed three times with the same buffer and heated at 95°C for 5 min in SDS-PAGE sample buffer. Coprecipitated full-length pol ß or pol ß fragments were analyzed by Western blotting using anti-pol ß Ab.

For immunoprecipitation, cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 400 mM NaCl, 25 mM NaF, 0.2% Triton X-100, 0.3% Nonidet P-40, and protease inhibitors (2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml bestatin)] for 20 min at 4°C. Lysates were centrifuged at 13,000 rpm for 30 min at 4°C, and the concentration of NaCl was adjusted to 80 mM in supernatants. The supernatants were incubated with the indicated antibodies and protein G-Sepharose overnight at 4°C. After being washed, samples were heated at 95°C for 5 min in SDS-PAGE sample buffer and used for further analysis.

In vitro methylation assay
Purified recombinant proteins were incubated with 1–5 µg of GST-PRMT1 in 30 µl of methylation buffer (50 mM HEPES (pH 8.0), 0.01% (v/v) Nonidet P-40, 10 mM NaCl, 1 mM DTT, and 1 mM PMSF) supplemented with 25 nCi of S-adenosyl-L-(methyl-14C) methionine (14C-SAM; Amersham Biosciences; radioactive methylation) or 20 nmol of S-adenosyl-L-methionine sulfate p-toluenesulfonate (SAMe-PTS, Sigma, St. Louis, MO) for 1 h at 30°C (cold methylation). Reactions were stopped either by adding 2x SDS-PAGE sample buffer, followed by heating at 95°C for 5 min or by adding of 100mM NaCl and incubating at 4°C for 10 min. Samples were analyzed by SDS-PAGE followed by autoradiography.

Mass spectrometry
Pol ß was methylated in vitro by GST-PRMT1 bound to glutathione sepharose beads and subsequently treated with chymotrypsin proteases. The peptide mixtures were separated on a C18 reversed-phase column with an inner diameter of 75 µm, which also served as the emitter for nanospray. The peptide solutions containing 1.0 pmol of digested pol ß were mixed with 5 µl of solvent A (5% acetonitrile and 0.2% formic acid) and loaded onto the capillary column. LC-MS/MS analysis was performed on an LCQ Deca XP ion trap mass spectrometer (Thermo Electron, San Jose, CA). Each full MS scan was followed by three MS/MS scans of the most intense ion signals detected in the MS mode. The dynamic exclusion feature was enabled to maximize the detection of less abundant peptide ions. Sample loading, gradient delivery, and all the mass spectrometric scan functions were controlled by the Xcalibur software (Thermo Electron). CID spectra were searched against a human protein sequence database consisting of the respective datasets from UniProt (43) , by using the SEQUEST software tool. Search parameters included a peptide mass tolerance of ±2.0 dalton (Da) and a fragment ion mass tolerance of ±0.6 Da as well as provisions for both unmodified and oxidized methionines (+16). Methylation sites were found by setting "different Mods" parameter to 14 Da for mono-methylated arginines and to 28 Da for dimethylated arginines.

DNA polymerase assays
DNA polymerase reactions were performed in a final volume of 10 µl containing 50 mM Tris-HCl (pH 7.6), 0.25 mg/ml BSA, 1 mM DTT, 0.8 mM MnCl2, and 100 µM of each unlabeled dNTPs, 32P-5' end-labeled primer annealed to the template and the indicated amounts of pol ß. The reactions were incubated for 15 min at 37°C. Reactions were stopped by adding gel loading buffer [95% (v/v) formamide, and 20 mM EDTA, pH 8.0] and heating at 95°C for 5 min. Reaction products were resolved on a 10% polyacrylamide, 7 M urea sequencing gel, dried, and exposed to an X-ray film.

dRP-lyase assay
The dRP-lyase activity of methylated and nonmethylated pol ß was analyzed as described previously (41) including some modifications: 100 fmol of a 34-mer G/U substrate 32P-labeled at the 3' end of the uracil containing strand were first treated by UDG (50 fmol) and APE1 (2 fmol) for 10 min at 30°C in a reaction mixture containing 40 mM HEPES-KOH (pH 7.6), 70 mM KCl, 7 mM MgCl2, 1 mM DTT, and 500 µg/ml BSA. The resulting nicked product containing a 5'-dRP moiety was subsequently used as a substrate for pol ß. dRP-lyase activity of pol ß was measured by incubation of the specific substrate with the indicated amounts of methylated or control pol ß, for 10 min at 37°C. The reaction was stopped by adding 333 mM NaBH4 followed by an overnight incubation at 4°C to stabilize the remaining dRP moieties. The DNA was recovered by ethanol precipitation and resolved on a 15% polyacrylamide, 7 M urea sequencing gel, dried, and exposed to an X-ray film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pol ß forms a complex with PRMT1
To examine whether PRMTs interact with pol ß, we performed immunoprecipitations of transiently expressed myc-tagged PRMTs from total 293T cell extracts supplemented with recombinant pol ß. Immunoprecipitations with an anti-myc Ab or a control Ab and subsequent Western blotting using an anti-pol ß Ab revealed that pol ß formed a specific complex with PRMT1 (Fig. 1 A). To assess whether PRMT1 directly interacts with pol ß, recombinant purified GST-PRMT1 was bound to glutathione beads and incubated with recombinant purified full-length pol ß. After several washes with the incubation buffer, bound proteins were resolved by SDS-PAGE followed by immunoblot detection of pol ß. In this analysis, pol ß was able to directly bind to GST-PRMT1 but not to GST alone (Fig. 1B ).


Figure 1
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  		Figure 1. Pol ß forms a complex and interacts directly with PRMT1 through its dRP-lyase domain. A) Myc-PRMT1 was immunoprecipitated from 293T cell extracts supplemented with recombinant his-pol ß using antimyc Ab or a mouse Ab as control. Coimmunoprecipitated proteins were visualized by Western blotting using anti-pol ß Ab. B) GST-pull down experiment using pol ß and GST-PRMT1 or GST as a control. Interaction of pol ß with GST-PRMT1 was monitored by Western blotting using anti-pol ß Ab. C) Pol ß and deletion mutants were analyzed in GST pull-down experiment using GST-PRMT1 or GST as described in B.

The lyase domain of pol ß is required for the interaction with PRMT1
To further characterize the interaction of pol ß with PRMT1, three pol ß deletion mutants lacking structural and functional subdomains were cloned, expressed in E. coli, and purified as His-tagged proteins (Fig. 1C , inputs). GST pull-down experiments were performed with these mutants using GST-PRMT1 or GST as a control, and bound proteins were subsequently analyzed by using an anti-pol ß Ab. The analysis revealed that pol ß lacking the lyase domain was no longer able to bind to PRMT1 (Fig. 1C ). In contrast, the deletion of the thumb or thumb/palm regions of pol ß did not affect the interaction of pol ß with PRMT1. Together, these results indicate that the lyase domain of pol ß is required for the direct interaction with PRMT1.

Pol ß is methylated in vitro by PRMT1
Since PRMT1 and pol ß interacted directly, we investigated whether PRMT1 could methylate pol ß in vitro. Transiently expressed PRMT1 was immunoprecipitated from 293T cells and tested in an in vitro radioactive methylation assay for its ability to methylate pol ß. The results of the experiment indicated that PRMT1 efficiently methylated pol ß in vitro. The extent of pol ß methylation was comparable to that observed for core histones, which were used as a positive control for PRMT1-mediated methylation (Fig. 2 A). To further confirm that PRTM1 methylates pol ß, bacterially expressed and purified GST-PRMT1 was used in an in vitro methylation assay (Fig. 2B ). GST-PRMT1 methylated pol ß and core histones but did not methylate Fen1 and Lig1. Fen1 and Lig1 were used as negative controls to verify the specificity of pol ß methylation by PRMT1 (Fig. 2B and data not shown). Furthermore, methylation of pol ß was observed with baculovirus expressed and purified full-length PRMT1 but not PRMT4 (data not shown), indicating that PRMT1 was a specific methyltransferase for pol ß. Quantitation of pol ß methylation revealed that 40% of the pol ß molecules were methylated in the presence of a 10-fold molar excess of C14-S-adenosyl methionine (SAM).


Figure 2
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  		Figure 2. Pol ß is methylated in vitro by PRMT1. A) Overexpressed myc-tagged PRMT1 was immunoprecipitated and incubated with C14-SAM and histones (10 µg) or recombinant pol ß (5 µg). Proteins were resolved using a 15% SDS-PAGE, stained with Coomassie blue (left panel), dried, and subsequently analyzed by autoradiography (right panel). B) Purified GST-tagged PRMT1 (5 µg) was incubated with C14-SAM and histones (10 µg), pol ß (5 µg), or Fen1 (10 µg). Proteins were resolved using a 15% SDS-PAGE, stained with Coomassie blue (left panel), dried, and subsequently analyzed by autoradiography (right panel).

Identification of a PRMT1-dependent methylation site in pol ß
Methylated pol ß was digested with chymotrypsin, and the resulting peptides were analyzed by LC/MS/MS. For the methylated protein, one triply charged peptide [EDLRKNEDKLNHHQ(mono-metR137)IGL] was found with an Xcorr score over the threshold limit of 2.5. The measured ion mass of the peptide corresponded to the theoretical one. This experiment suggested that the arginine 137 was monomethylated by GST-PRMT1. The identified arginine residue is located at the C-terminal end of the polymerase finger subdomain within the putative PCNA binding motif I. To confirm this methylation site, the arginine was replaced with a lysine by site-directed mutagenesis. This substitution allows to maintain the positive charge of the amino acid and to minimize changes in the local environment of the protein. Wild-type (WT) or mutated pol ß harboring an R137K mutation was expressed and purified from E. coli. Subsequently, all proteins were subjected to in vitro methylation by GST-PRMT1. Methylation of pol ß mutated at R137 was substantially reduced, indicating that arginine 137 was a target for GST-PRMT1. However, residual methylation of the mutant pol ß suggests the presence of additional PRMT1 target sites that remain to be elucidated (Fig. 3 A).


Figure 3
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  		Figure 3. Pol ß is methylated in vitro and in vivo by PRMT1 at R137. A) Equimolar amounts of WT pol ß and mutant pol ß R137K were methylated by GST-PRMT1. Proteins were resolved using a 12% SDS-PAGE, stained with Coomassie blue (upper panel), dried, and subsequently analyzed by autoradiography (lower panel). B) Methylation of pol ß by PRMT1 in vivo. 293T cells were labeled 24 h after transfection with L-[methyl-3H]-methionine as described in Materials and Methods. Recombinant HA-tagged pol ß and myc-tagged PRMT1 were immunoprecipitated from the cell lysates and analyzed by SDS-PAGE (lower panel) followed by autoradiography (upper panel). C) Expression of HA-pol ß and myc-PRMT1 was analyzed in total cell lysates by Western blotting using anti-hemagglutinin and antimyc antibodies, respectively. D) HA-pol ß or HA-pol ß R137K was immunoprecipitated from 293T cells expressing recombinant myc-PRMT1 using anti-hemagglutinin Ab. Coimmunoprecipitated proteins were visualized by Western blotting using antimyc Ab. E) Expression of recombinant proteins shown in D was analyzed by Western blotting using indicated antibodies.

Pol ß is methylated in vivo by PRMT1
Next, we investigated whether R137 of pol ß is a major target for methylation by PRMT1 in vivo. WT pol ß or R137K mutant was cotransfected with PRMT1 in 293T cells. The cells were metabolically labeled with L-[methyl-3H]methionine for 3.5 h in the presence of translation inhibitors, as described previously (42) . Recombinant pol ß was immunoprecipitated with an anti-hemagglutinin Ab, separated by SDS-PAGE, and analyzed by autoradiography (Fig. 3B ). Expression of recombinant proteins was controlled by Western blotting using the indicated antibodies (Fig. 3C ). In vivo activity of recombinant PRMT1 expressed in 293T cells was monitored as automethylation of the enzyme (Fig. 3B ). The methylation of WT pol ß was substantially increased in cells overexpressing PRMT1 when compared to that in cells transfected with pol ß alone. We could not detect such an increase in methylation when the R137K mutant of pol ß was analyzed under similar conditions. This suggests that R137 residue represents a major PRMT1 methylation site in pol ß in vivo. To exclude that R137K mutation may affect the interaction between PRMT1 and pol ß, we compared the ability of WT and mutated pol ß to interact with PRMT1 in vivo (Fig. 3D, E ). Both recombinant proteins, WT pol ß and R137K mutant, were able to coimmunoprecipitate PRMT1 from 293T cells with similar efficiency. These results confirmed that the observed defect in methylation of R137K mutant by PRMT1 was due to the absence of methylation site in pol ß. Together, these results indicate that pol ß is an in vivo substrate for arginine methylation by PRMT1.

Methylation of pol ß by PRMT1 does not affect its DNA polymerase and dRP-lyase activities
Because PRMT1 specific methylation was mapped to the polymerase domain of pol ß, we investigated whether R137 methylation may affect polymerase activity of the enzyme. Different amounts of WT or R137K mutant of pol ß were methylated by GST-PRMT1 and subsequently tested in a DNA polymerase assay using as a substrate a 32P-labeled 17mer oligonucleotide annealed to a 73mer oligonucleotide (Fig. 4 A). Comparison of the products synthesized by methylated and nonmethylated WT or mutant pol ß revealed that methylation did not significantly affect the polymerase activity of pol ß.


Figure 4
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  		Figure 4. Methylation of pol ß by PRMT1 does not affect its DNA polymerase and dRP-lyase activities. A) Titration of pol ß (1, 3, 6 and 25 ng) in a polymerase assay using a 17/73 mer DNA template. Comparison of GST-PRMT1 methylated vs. nonmethylated forms of pol ß is shown. WT pol ß or mutant pol ß R137K were included in this assay. B) Titration of pol ß (0.004, 0.4, 4, and 40 pg) in a dRP-lyase assay using a G/U mismtach template treated by UDG and APE1. Comparison of GST-PRMT1 methylated vs. nonmethylated pol ß. First lane shows G/U mismatch substrate treated with UDG.

Next, we examined the effect of R137 methylation on the dRP-lyase activity of pol ß (Fig. 4B ). Different amounts of methylated or nonmethylated pol ß were tested in a dRP-lyase assay as described in Materials and Methods. Methylation of pol ß did not result in any significant changes in its dRP-lyase activity (Fig. 4B ). Thus, our data indicate that methylation of R137 is not involved in the regulation of the DNA polymerase and dRP-lyase activities of pol ß.

Methylation of pol ß abolishes its binding to PCNA
As mentioned above, R137 is located within the PCNA binding motif I. This prompted us to investigate whether the methylation of pol ß could affect its ability to bind PCNA. Pol ß was methylated by GST-PRMT1 and subsequently tested in a GST pull down assay with PCNA. In this experiment, recombinant pol ß interacted with GST-PCNA bound to glutathione sepharose beads. Under similar conditions, GST alone was not able to pull down pol ß, suggesting the specificity of the PCNA/pol ß interaction (Fig. 5 A). Next, we conducted GST pull down assays using pol ß metylated by PRMT1. The results of the experiments indicated that methylation of pol ß by PRMT1 strongly reduced the interaction between PCNA and pol ß under the tested conditions (Fig. 5A ). Nonspecific effects, which could be due to the presence of SAM and PRMT1 in the reaction mixtures, were ruled out by control experiments (Fig. 5A and data not shown). These findings were further confirmed by in vivo studies. The hemagglutinin (HA)-tagged WT pol ß or R137K mutant were coexpressed with PCNA in 293T cells. Recombinant proteins were immunoprecipitated with an anti-hemagglutinin Ab, separated by SDS-PAGE, and analyzed by Western blotting with an anti-PCNA Ab (Fig. 5B, C ). Mutation of R137 to lysine increased the amount of PCNA that was coimmunoprecipitated with pol ß, strengthening the importance of R137 for interaction with PCNA. These results are consistent with a negative effect of R137 methylation on the ability of pol ß to interact with PCNA in vitro. These data suggest that methylation of pol ß by PRMT1 might regulate the binding of pol ß to PCNA.


Figure 5
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  		Figure 5. PRMT1-mediated methylation of pol ß abolishes its binding to PCNA. A) GST-pull down of methylated or nonmethylated pol ß using GST-PCNA. Pol ß was detected by Western blotting using an anti-pol ß Ab. To control the effect of SAM on pol ß/PCNA interaction, methylated and nonmethylated pol ß was supplemented with SAM and checked for interaction with GST-PCNA. B) Coimmunoprecipitation of HA-pol ß or HA-pol ß R137K with PCNA transiently expressed in 293T cells. Recombinant proteins were immunoprecipitated with an anti-hemagglutinin Ab and analyzed by Western blotting using anti-PCNA or anti-hemagglutinin antibodies. C) Expression of PCNA was analyzed in total cell extracts by Western blotting using an anti-PCNA Ab.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PCNA is a well-known component of the DNA replication system in mammalian cells and plays essential roles in many aspects of DNA metabolism by mediating interactions of proteins with DNA (44) . Additionally, PCNA participates in DNA repair, including BER, nucleotide excision repair, and mismatch repair, as well as in the control of cell cycle, apoptosis, and transcription (45 , 46) . Thus, PCNA has been termed a "cellular communicator" by its virtue to connect various cellular processes (47) . PCNA does not possess any known enzymatic activity, and the cellular functions of PCNA are achieved mainly due to the ability of this protein to directly interact with a broad range of other cellular proteins. Pol ß was identified as an interacting partner for PCNA (20) . The interaction was confirmed by cell extract immunoprecipitation and yeast two-hybrid experiments. Kedar et al. found that the proteins interacted directly and mapped the region of pol ß responsible for its interaction with PCNA to a sequence resembling a consensus PIM (20) , without providing a functional relevance of this interaction.

In our study, we provide first evidence that interaction of pol ß with PCNA is regulated by PRMT1-mediated methylation of pol ß. We report that the mammalian pol ß forms a complex with PRMT1. A direct physical interaction was confirmed with bacterially expressed and purified pol ß fragments. Furthermore, we found that pol ß was methylated in vitro and in vivo by PRMT1 and identified arginine 137 as a site of PRMT1 methylation. Investigation of the biochemical consequence of pol ß methylation revealed that this modification specifically abolished interaction of pol ß with PCNA.

Many of the PCNA-binding proteins contain a conserved PCNA interacting motif or PIM (47) . Three sequences resembling the consensus PIM (PIM I, II, and III) were previously identified in the polymerase domain of pol ß (20) . Mutation of the PIM-like sequence II resulted in a reduced affinity of pol ß binding to PCNA, supporting the relevance of this motif. Our results suggest that in addition to PIM II, the PIM-like sequence I strongly contributes to the PCNA/pol ß interaction. Analysis of X-ray structure of pol ß (48 , 49) indicated that similar to PIM II, PIM I is also exposed on the surface of pol ß molecule allowing an easy access for PCNA. Moreover, we found that the PRMT1-mediated methylation of R137 residue located within the PIM I of pol ß reduced its binding to PCNA. Although the methylation of additional arginine residues in pol ß by PRMT1 cannot be currently excluded, our data provide evidence that the regulation of PCNA/pol ß interaction is primarily modulated by methylation of PIM I. Consistent with this, the sequence analyses revealed that human pol ß, used in this study, does not contain arginine residues within the two other PIM-like sequences II and III. This minimizes the possibility that methylation of other arginines in pol ß may interfere with the PCNA interaction.

The physiological relevance of PCNA/pol ß interaction remains largely unknown. However, the regulated nature of pol ß binding to PCNA, shown in this study, may suggest a regulatory function of this interaction in pol ß-dependent processes. One can imagine that PCNA/pol ß complex could coordinate the functions of pol ß and other PCNA-binding proteins involved in BER, e.g., Fen1 and Lig I (44) . This hypothesis is supported by earlier findings that PCNA enhances pol ß-dependent LP-BER of AP sites by stimulation of Fen1 activity (9) . Alternatively, PCNA may regulate pol ß functions by facilitating its recruitment to DNA/protein complexes formed during BER. PCNA is known to anchor various proteins onto DNA and functions in vitro as a processivity factor for several DNA polymerases including pol {delta} and pol {epsilon} (50) . More studies are required to clarify the particular role of the regulated pol ß/PCNA interaction in the context of BER.

The role of protein arginine methylation in DNA damage response and DNA repair just starts to emerge. PRMT1 has recently been described to methylate DNA damage checkpoint protein Mre11. Mutation of the methylation sites severely impaired the exonuclease activity of Mre11 (38) . Moreover, cells containing hypomethylated Mre11 displayed intra-S-phase DNA damage checkpoint defects (38) due to the compromised function of MRN complex. Another DNA-damage response protein 53BP1 is also arginine methylated by PRMT1. Methylation of 53BP1 regulates its DNA binding activity and recruitment to the {gamma}-H2AX foci associated with sites of DNA breaks (51) . Recently, we have provided evidence that PRMT6 plays a direct role in the regulation of BER by methylating pol ß at R83 and 152 and promoting its DNA repair functions (42) .

In this study, we found that the dRP-lyase domain of pol ß was strictly required for the direct interaction with PRMT1 whereas deletion of thumb or thumb/palm regions in pol ß did not interfere with the PRMT1 binding. We have previously reported that dRP-lyase domain mediated the pol ß/PRMT6 interaction. It remains to be investigated whether PRMT1 and PRMT6 interact with the same or different regions of the pol ß polypeptide. It is also important to investigate whether PCNA or other pol ß interacting proteins can modulate methylation of pol ß by PRMT1. We have demonstrated that PRMT1, associated with pol ß, specifically mono-methylated R137 in pol ß. PRMT1 is known to methylate many different proteins within glycine- and arginine-rich regions, referred as ‘GAR’ motifs. These clustered basic motifs are especially abundant in proteins involved in RNA metabolism. The context of methylated residues suggested the importance of residues in the –1 to +2 positions for recognition of the substrate arginine (52) and a consensus sequence surrounding actual methylation sites was proposed as followed: (F/G)GGR(met)GG(G/F). Arginine 137, identified as the PRMT1 methylation site in pol ß, does not follow this rule but is the only arginine in the predicted PCNA interaction motif. Since the physiological conditions under which pol ß is methylated by PRMT1 in the cells are not know, it is difficult to assign a specific function to this modification in vivo. However, one of the clear consequences of PRMT1 mediated methylation was abolishment of PCNA/pol ß interaction. Therefore, we propose that methylation of pol ß by PRMT1 may play a regulatory role in BER by preventing the participation of pol ß in PCNA-dependent DNA metabolic events.

In conclusion, our findings strengthen the hypothesis that PRMT1, as already shown for PRMT6, plays an essential role in DNA BER through the direct interaction and modification of the main BER enzyme pol ß.


   ACKNOWLEDGMENTS
 
We thank M. T. Bedford (University of Texas, USA) for providing PRMT1 construct and P. Schär for the helpful advice. We are grateful to all the members of the Institute of Veterinary Biochemistry and Molecular Biology (University of Zurich, Switzerland) for their advices and discussions. This work was supported in part by the Swiss National Science Foundation program 31–109312.05, 31–109315.05, and 3339C0–101584/1. P. O. Hassa, U. Hübscher, and M. O. Hottiger are supported by the Kanton of Zurich.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication March 22, 2006. Accepted for publication July 24, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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