Requirements for the Interaction of Mouse Polκ with Ubiquitin and Its Biological Significance
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m709275200
ISSN1083-351X
AutoresCaixia Guo, Tie-Shan Tang, Magda Bienko, Ivan Đikić, Errol C. Friedberg,
Tópico(s)Cancer-related Molecular Pathways
ResumoPolκ protein is a eukaryotic member of the DinB/Polκ branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polκ in this process has remained unknown. Here we report that mouse Polκ can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polκ with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polκ. In addition, Polκ binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polκ. The ubiquitin-binding motifs are also required for Polκ to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polκ half-life. Finally, we have examined levels of Polκ expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation. Polκ protein is a eukaryotic member of the DinB/Polκ branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polκ in this process has remained unknown. Here we report that mouse Polκ can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polκ with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polκ. In addition, Polκ binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polκ. The ubiquitin-binding motifs are also required for Polκ to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polκ half-life. Finally, we have examined levels of Polκ expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation. Translesion DNA synthesis (TLS) 3The abbreviations used are: TLStranslesion DNA synthesisUBZubiquitin-binding zinc finger motifUbubiquitinCHXcycloheximideGSTglutathione S-transferasePCNAproliferating cell nuclear antigenBPDEbenzo-[a]pyrene-dihydrodiol epoxidemPolκmouse PolκhPolκhuman PolκEGFPenhanced green fluorescent proteinHAhemagglutininMEFmouse embryonic fibroblast. is one of several biochemical mechanisms by which cells can tolerate DNA damage that arrests semiconservative DNA synthesis (1Friedberg E.C. Nat. Rev. Mol. Cell Biol. 2005; 6: 943-953Crossref PubMed Scopus (212) Google Scholar, 2Friedberg E.C. Lehmann A.R. Fuchs R.P. Mol. Cell. 2005; 18: 499-505Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). This process requires the action of specialized DNA polymerases present in bacteria (such as Escherichia coli), lower eukaryotes, and vertebrates. Lower eukaryotes, particularly vertibrates, contain multiple such enzymes, suggesting the ability to bypass many types of DNA damage. translesion DNA synthesis ubiquitin-binding zinc finger motif ubiquitin cycloheximide glutathione S-transferase proliferating cell nuclear antigen benzo-[a]pyrene-dihydrodiol epoxide mouse Polκ human Polκ enhanced green fluorescent protein hemagglutinin mouse embryonic fibroblast. Several specialized DNA polymerases are members of a novel polymerase family, the Y-family (3Ohmori H. Friedberg E.C. Fuchs R.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). These enzymes are devoid of 3′ → 5′ proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (4Bebenek K. Kunkel T.A. Adv. Protein Chem. 2004; 69: 137-165Crossref PubMed Scopus (248) Google Scholar). Members of this family in mammalian cells include Polκ, Polι, and Polη, all of which can extend primers for varying distances past various types of template damage (4Bebenek K. Kunkel T.A. Adv. Protein Chem. 2004; 69: 137-165Crossref PubMed Scopus (248) Google Scholar). A fourth member of the Y-family, REV1 protein, is able to catalyze the incorporation of only one or two dCMP moieties, regardless of the template base composition (5Lawrence C.W. Adv. Protein Chem. 2004; 69: 167-203Crossref PubMed Scopus (116) Google Scholar). Polκ, Polι, and Polη have been shown to interact with REV1 protein via a highly conserved C-terminal domain in REV1 (6Guo C. Fischhaber P.L. Luk-Paszyc M.J. Masuda Y. Zhou J. Kamiya K. Kisker C. Friedberg E.C. EMBO J. 2003; 22: 6621-6630Crossref PubMed Scopus (302) Google Scholar, 7Ohashi E. Murakumo Y. Kanjo N. Akagi J. Masutani C. Hanaoka F. Ohmori H. Genes Cells. 2004; 9: 523-531Crossref PubMed Scopus (222) Google Scholar, 8Tissier A. Kannouche P. Reck M.P. Lehmann A.R. Fuchs R.P. Cordonnier A. DNA Repair. 2004; 3: 1503-1514Crossref PubMed Scopus (178) Google Scholar). These polymerases also interact with PCNA (9Lehmann A.R. Niimi A. Ogi T. Brown S. Sabbioneda S. Wing J.F. Kannouche P.L. Green C.M. DNA Repair. 2007; 6: 891-899Crossref PubMed Scopus (322) Google Scholar, 10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), and recent observations suggest that PCNA plays a key role in promoting the access of specialized polymerases to arrested replication forks (11Kannouche P.L. Wing J. Lehmann A.R. Mol. Cell. 2004; 14: 491-500Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar, 12Prakash S. Johnson R.E. Prakash L. Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (834) Google Scholar, 13Ulrich H.D. Cell Cycle. 2004; 3: 15-18Crossref PubMed Google Scholar, 14Stelter P. Ulrich H.D. Nature. 2003; 425: 188-191Crossref PubMed Scopus (689) Google Scholar, 15Watanabe K. Tateishi S. Kawasuji M. Tsurimoto T. Inoue H. Yamaizumi M. EMBO J. 2004; 23: 3886-3896Crossref PubMed Scopus (457) Google Scholar). Disruption of the PolK gene in mouse and chicken cells results in significant sensitivity to killing by benzo[a]pyrene-dihydrodiol epoxide (BPDE) and UV radiation (16Ogi T. Shinkai Y. Tanaka K. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15548-15553Crossref PubMed Scopus (200) Google Scholar, 17Schenten D. Gerlach V.L. Guo C. Velasco-Miguel S. Hladik C.L. White C.L. Friedberg E.C. Rajewsky K. Esposito G. Eur. J. Immunol. 2002; 32: 3152-3160Crossref PubMed Scopus (126) Google Scholar, 18Okada T. Sonoda E. Yamashita Y.M. Koyoshi S. Tateishi S. Yamaizumi M. Takata M. Ogawa O. Takeda S. J. Biol. Chem. 2002; 277: 48690-48695Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Polκ-deficient mouse embryonic stem and fibroblast cells also show moderate sensitivity to methyl methanesulfonate (19Takenaka K. Ogi T. Okada T. Sonoda E. Guo C. Friedberg E.C. Takeda S. J. Biol. Chem. 2006; 281: 2000-2004Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Consistent with these results, primer extension assays have shown that human Polκ can support TLS across sites of base loss, acetylaminofluorene-G adducts, benzo[a]pyrene-G adducts, and thymine glycol (4Bebenek K. Kunkel T.A. Adv. Protein Chem. 2004; 69: 137-165Crossref PubMed Scopus (248) Google Scholar). However, the enzyme does not support primer extension past thymine-thymine (T T) dimers or [6,4]pyrimidine-pyrimidone photoproducts (4Bebenek K. Kunkel T.A. Adv. Protein Chem. 2004; 69: 137-165Crossref PubMed Scopus (248) Google Scholar). Similar to DNA polymerase Polξ, Polκ is also efficient in extending sites of replicative bypass by other specialized polymerases during TLS, at least in vitro (12Prakash S. Johnson R.E. Prakash L. Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (834) Google Scholar). Furthermore, overexpression of Polκ in mammalian cells promotes pleiotropic genetic alterations and tumorigenesis (20Bavoux C. Hoffmann J.S. Cazaux C. Biochimie (Paris). 2005; 87: 637-646Crossref PubMed Scopus (41) Google Scholar, 21Bavoux C. Leopoldino A.M. Bergoglio V. O-Wang J. Ogi T. Bieth A. Judde J.G. Pena S.D. Poupon M.F. Helleday T. Tagawa M. Machado C. Hoffmann J.S. Cazaux C. Cancer Res. 2005; 65: 325-330PubMed Google Scholar). The relaxed fidelity of Polκ renders it error prone when copying undamaged DNA. Hence, access of the enzyme to sites of undamaged DNA must be tightly regulated to avoid mutational catastrophes. It has been reported that Polκ accumulates in microscopically discrete nuclear foci in UV radiation- or BPDE-treated cells (22Ogi T. Kannouche P. Lehmann A.R. J. Cell Sci. 2005; 118: 129-136Crossref PubMed Scopus (67) Google Scholar, 23Bergoglio V. Bavoux C. Verbiest V. Hoffmann J.S. Cazaux C. J. Cell Sci. 2002; 115: 4413-4418Crossref PubMed Scopus (51) Google Scholar, 24Bi X. Slater D.M. Ohmori H. Vaziri C. J. Biol. Chem. 2005; 280: 22343-22355Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In addition, the C-terminal 97 amino acids of Polκ, which include a C2HC zinc finger, a bipartite nuclear localization signal, and a putative PCNA binding site, are important for the localization of Polκ in nuclear foci (22Ogi T. Kannouche P. Lehmann A.R. J. Cell Sci. 2005; 118: 129-136Crossref PubMed Scopus (67) Google Scholar). However, the underlying mechanism of TLS by Polκ and other specialized DNA polymerases remains unclear. To further our understanding of the role of Polκ in TLS and in spontaneous and DNA damage-associated mutagenesis, we searched for proteins that interact with mouse Polκ by screening a mouse testis cDNA library using the yeast two-hybrid system (6Guo C. Fischhaber P.L. Luk-Paszyc M.J. Masuda Y. Zhou J. Kamiya K. Kisker C. Friedberg E.C. EMBO J. 2003; 22: 6621-6630Crossref PubMed Scopus (302) Google Scholar). We report here that ubiquitin binds strongly to Polκ bait protein. We examined the interaction of ubiquitin with Polκ and show that this interaction requires two novel zinc fingers, called ubiquitin-binding motifs (UBZs) (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar), resident in the C-terminal half of Polκ. We also demonstrate that Polκ binds monoubiquitinated PCNA more robustly than nonubiquitinated PCNA. The UBZs are required for Polκ to form nuclear foci after UV radiation. We measured the half-life of endogenous Polκ as 5.4 h and show that mutational disruption of the UBZs does not alter the half-life of Polκ protein. Finally, we examined levels of Polκ expression following exposure of mouse cells to BPDE or UVB radiation. Plasmids–For yeast two-hybrid screening, pGBT9/mouse PolK plasmid was cloned as described (6Guo C. Fischhaber P.L. Luk-Paszyc M.J. Masuda Y. Zhou J. Kamiya K. Kisker C. Friedberg E.C. EMBO J. 2003; 22: 6621-6630Crossref PubMed Scopus (302) Google Scholar). For binding assays, full-length mouse PolK cDNA was cloned in pCMV-Myc or pCMV-HA (Clontech) to generate Myc or HA fusion proteins. For confocal study, mouse PolK cDNA with the first ATG codon deleted was PCR-amplified and cloned in the SalI site of pEGFP-C3 (Clontech) to generate an EGFP fusion protein. Human POLK cDNA was PCR-amplified and cloned in the BamHI site of pEGFP-C1 (Clontech). Isolated UBZs of mPolK were PCR-amplified and cloned into pCMV-Myc (Clontech) or pGEX4T-2 vectors (Amersham Biosciences). Ubiquitin, PCNA, and PCNA-ubiquitin chimera (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar) were subcloned in pGEX4T-2 to produce GST fusion proteins as reported (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar). A series of mutant mPolK constructs was generated using the QuikChange site-directed mutagenesis kit (Stratagene). Ubiquitin was cloned in pcDNA3-HA as described (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar). Yeast Two-hybrid Assay–The pGBT9/mouse PolK plasmid was used to screen a mouse testis cDNA library as described (6Guo C. Fischhaber P.L. Luk-Paszyc M.J. Masuda Y. Zhou J. Kamiya K. Kisker C. Friedberg E.C. EMBO J. 2003; 22: 6621-6630Crossref PubMed Scopus (302) Google Scholar). Cell Culture and Treatments–COS7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For transient transfection experiments COS7 and HEK293T (human embryonic kidney) cells were transfected with the indicated constructs using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Cells were harvested for further analysis 48 h after transfection. The SV40-transformed human fibroblast MRC5 was kindly provided by Dr. Alan R. Lehmann, University of Sussex. MRC5 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection and UV irradiation were carried out as described previously (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Nuclear Protein Extraction and Western Blotting–Wild-type MEFs were prepared and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as described (17Schenten D. Gerlach V.L. Guo C. Velasco-Miguel S. Hladik C.L. White C.L. Friedberg E.C. Rajewsky K. Esposito G. Eur. J. Immunol. 2002; 32: 3152-3160Crossref PubMed Scopus (126) Google Scholar). Treatments using genotoxic agents were as follows. 1), BPDE (NCI National Institutes of Health carcinogen repository) was dissolved in dimethyl sulfoxide. 1 mm BPDE was added to exponentially growing cells and incubated for 1 h, and the cells were washed twice with phosphate-buffered saline and incubated with fresh medium. 2), UVB radiation at 25 J/m2 was performed in a UV cross-linker (UV Stratalinker 2400, Stratagene). Nuclear extracts were harvested as described previously (27Guo C. Gao T. Confer N. Velasco-Miguel S. Friedberg E.C. DNA Repair. 2005; 4: 397-402Crossref PubMed Scopus (11) Google Scholar) at different time points. Protein Half-life Determination–COS7 cells were transfected with the wild-type and UBZs mutant HA-Polκ constructs. Twenty-four h later, the transfected cells were aliquoted into 8–10 35-mm dishes to continue to culture for ∼16 h. The half-life of Polκ was determined by treating cells with 25 μg/ml cycloheximide (CHX) (Sigma) for 0–7 h to inhibit protein synthesis and then preparing cell lysates to determine Polκ levels by Western blotting. For endogenous Polκ, wild-type MEFs were treated with 25 μg/ml CHX for 0–9 h. The content of Polκ and β-actin bands was quantified by Photoshop histogram. To calculate the half-life of Polκ protein, the content of the Polκ Western blot bands at each time point was normalized to the control (0 h) Polκ content and also refers to the β-actin content. The normalized data from several independent experiments were averaged together and semi-log plots were generated in Origin 4.1. Linear regression was performed, and the half-life was calculated from the fitted line equation. Antibodies–Rabbit polyclonal anti-HA and mouse monoclonal anti-HA and anti-Myc were purchased from Covance. Anti-FLAG M2 agarose affinity gel and anti-FLAG M2 monoclonal antibodies were purchased from Sigma. Hamster polyclonal antiserum against mouse Polκ was made by our laboratory (27Guo C. Gao T. Confer N. Velasco-Miguel S. Friedberg E.C. DNA Repair. 2005; 4: 397-402Crossref PubMed Scopus (11) Google Scholar). Rabbit polyclonal antiserum against mouse Polκ was generated with a 14-amino acid peptide (CNYLKIDTPRQEANE) containing an N-terminal cysteine residue conjugated with keyhole limpet hemocyanin as described (28Velasco-Miguel S. Richardson J.A. Gerlach V.L. Lai W.C. Gao T. Russell L.D. Hladik C.L. White C.L. Friedberg E.C. DNA Repair. 2003; 2: 91-106Crossref PubMed Scopus (72) Google Scholar). Anti-PCNA antibodies were purchased from Santa Cruz Biotechnology. Lysate Preparation, Co-immunoprecipitation, and Western Blotting–COS7 cells were transfected with pCMV-HA-mPolκ and pCMV5-FLAG-Ub. Harvested cell lysates were immunoprecipitated with anti-FLAG antibodies. HEK293T cells were transfected with pCMV-Myc-mPolκ and pcDNA3-HA-Ub. Harvested cell lysates were immunoprecipitated with anti-Myc antibodies. Immunoprecipitation and immunoblotting were performed as described (26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar). MRC cells were transfected with HA-mPolκ, and 40 h later they were UV-irradiated (25 J/m2). They were then incubated for 7 h prior to Triton extraction and cross-linking. Triton-insoluble proteins were solubilized and immunoprecipitated with anti-PCNA as described (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). GST Pulldown Assay–GST fusion proteins were expressed and purified on glutathione-agarose (Sigma) as described (6Guo C. Fischhaber P.L. Luk-Paszyc M.J. Masuda Y. Zhou J. Kamiya K. Kisker C. Friedberg E.C. EMBO J. 2003; 22: 6621-6630Crossref PubMed Scopus (302) Google Scholar). Purified mPolκ was pulled down by GST-Ub as described previously (26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar). For interaction between truncated/mutant mPolκ and GST-PCNA or GST-Ub constructs, transfected COS7/MRC5 cells were lysed with HEPES buffer and incubated with equal amounts of GST fusion proteins as described previously (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar). Samples were separated by SDS-PAGE and detected by immunoblotting with polyclonal antibodies against mPolκ or with monoclonal antibodies against Myc (9E10), HA (16B12), or PCNA. Immunofluorescence Microscopy–MRC5 cells were transfected using a panel of mutated/truncated EGFP-mPolκ and EGFP-hPolκ constructs and cultured for ∼40 h. They were then UV-irradiated and processed for immunofluorescence as described previously (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Images were acquired using a Nikon Eclipse TE2000-U confocal laser scanning microscope and processed using Adobe Photoshop 7.0. A minimum of 200 nuclei were analyzed for each construct and treatment. Mouse Polκ Interacts with Ubiquitin via Two Ubiquitin-binding Zinc Finger Domains and Undergoes Ubiquitination in Vivo–By screening a mouse testis cDNA library using mouse Polκ as bait, we identified both ubiquitin B and REV1 as interacting moieties. Because ubiquitin B is a polypeptide containing four tandem ubiquitin moieties, we anticipated that Polκ would also bind to monoubiquitin. This was confirmed by GST pull-down using purified Polκ and GST-ubiquitin (Fig. 1A). We observed that mouse Polκ protein, in addition to binding ubiquitin, undergoes monoubiquitination in vivo. HA-Polκ and FLAG-ubiquitin were expressed in COS7 cells, and cell lysates were immunoprecipitated with anti-FLAG antibodies. Not surprisingly, HA-Polκ protein was detected in the immunoprecipitate, reflecting its interaction with ubiquitin (Fig. 1B). However, an additional slower migrating band was reproducibly observed, suggesting the presence of monoubiquitinated HA-Polκ protein (Fig. 1B). This was directly confirmed by immunoprecipitation of Polκ from cells cotransfected with Myc-Polκ and HA-Ub followed by immunoblotting with anti-HA antibodies (Fig. 1C). Sequence analysis revealed that the duplicated C2HC zinc cluster domains in mouse Polκ (29Gerlach V.L. Aravind L. Gotway G. Schultz R.A. Koonin E.V. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11922-11927Crossref PubMed Scopus (190) Google Scholar) are in fact novel ubiquitin-binding domains called UBZs (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar). The mouse UBZs (each ∼30 amino acids in length) are located between amino acid residues 608 and 800 (Fig. 2A). To determine whether the UBZs in Polκ are required for binding ubiquitin, we incubated a fragment of Polκ bearing just the two UBZs (UBZ1 + UBZ2) with GST-ubiquitin and confirmed the interaction (Fig. 2B). To further document the requirement of the UBZs in Polκ for its interaction with ubiquitin, we generated a series of mutant constructs that deleted the N-terminal UBZ (UBZ1) (Polκ-UBZ1Δ), the C-terminal UBZ (UBZ2) (Polκ-UBZ2Δ), or both (Polκ-UBZΔ). Additionally, we generated constructs in which the amino acids Asp-642 and/or Asp-784 were mutated to Ala (D642A in UBZ1*, D784A in UBZ2*, D642A and D784A in UBZ*). Deletion of either UBZ significantly impaired binding to GST-ubiquitin (Fig. 2C), and deletion of both UBZs completely eliminated the interaction (Fig. 2C). Similar results were obtained when selected amino acids in the Polκ UBZs were mutated to alanine (Fig. 2D). Mutational inactivation of the UBZs in Polκ also impaired its monoubiquitination (Fig. 2E), and their deletion completely abolished monoubiquitination of Polκ (Fig. 2E). Collectively, these results suggest that the Polk UBZs are required for interaction between Polκ and ubiquitin and for monoubiquitination of the polymerase. The UBZs Are Required for Enhanced Association between Polκ and Monoubiquitinated PCNA–Recent studies have demonstrated that monoubiquitination of PCNA in cells exposed to UV radiation promotes a more robust interaction of this accessory replication protein with Polη, Polι, and REV1 protein (10Guo C. Sonoda E. Tang T.S. Parker J.L. Bielen A.B. Takeda S. Ulrich H.D. Friedberg E.C. Mol. Cell. 2006; 23: 265-271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 11Kannouche P.L. Wing J. Lehmann A.R. Mol. Cell. 2004; 14: 491-500Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar, 25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar, 30Plosky B.S. Vidal A.E. Fernandez de Henestrosa A.R. McLenigan M.P. McDonald J.P. Mead S. Woodgate R. EMBO J. 2006; 25: 2847-2855Crossref PubMed Scopus (176) Google Scholar). To determine whether an enhanced association also exists between Polκ and monoubiquitinated PCNA, we examined their interaction by GST pulldown experiments (26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar). Consistent with results shown previously (31Bi X. Barkley L.R. Slater D.M. Tateishi S. Yamaizumi M. Ohmori H. Vaziri C. Mol. Cell. Biol. 2006; 26: 3527-3540Crossref PubMed Scopus (145) Google Scholar), the interaction of purified Polκ with PCNA-Ub was more robust than with native PCNA (Fig. 3A). To determine whether the enhanced interaction is mediated via the UBZ domains, we incubated cell lysates expressing wild-type Polκ or those carrying mutations in the UBZ domains with GST-PCNA fusion proteins. As shown in Fig. 3B, the enhanced association with GST-PCNA-Ub was not observed with UBZ mutant preparations. To further support the result, HA-Polκ and its UBZ deletion derivatives were expressed in cells exposed to UV radiation to generate monoubiquitinated PCNA. The chromatin fraction was then isolated and immunoprecipitated with PCNA antibodies. Consistent with the results shown above, the amount of precipitated wild-type, but not UBZ-deleted, Polκ was significantly increased after UVC treatment (Fig. 3C). Interestingly, the level of UBZ-deleted Polκ in chromatin fractions was significantly reduced after UVC treatment (Fig. 3C). We isolated the chromatin fraction from wild-type cells after UVC treatment and immunoprecipitated it with anti-PCNA antibodies. Consistent with the results shown above, the amount of precipitated endogenous Polκ was significantly increased after UVC treatment (Fig. 3D). In summary, the results of the experiments reported thus far indicate that mouse Polκ can interact with ubiquitin in vitro, an interaction that requires functional UBZs, and that Polκ can itself undergo monoubiquitination. A robust association between monoubiquitinated PCNA and Polκ also requires functional UBZs. Polκ UBZs Are Required for Association of Polκ with Replication Factories in Cells Exposed to UV Radiation–To validate the results described above in living cells, we transfected wild-type and UBZ-deleted EGFP-mouse Polκ constructs into fibroblasts. We observed strict nuclear localization of EGFP-mouse Polκ protein, regardless of the presence or absence of the UBZs (Fig. 4A). As reported previously for human Polκ (22Ogi T. Kannouche P. Lehmann A.R. J. Cell Sci. 2005; 118: 129-136Crossref PubMed Scopus (67) Google Scholar), in ∼4% of cells transfected with wild-type EGFP-mouse Polκ the protein was concentrated in nuclear foci (Fig. 4A). When cells transfected with EGFP-mouse Polκ were exposed to UV radiation and incubated for 8–16 h, the fraction of cells with discrete nuclear foci increased to ∼55.3% (Fig. 4B). Interestingly, the number of cells with mouse Polκ foci was higher than that observed when cells were transfected with human Polk and exposed to UV radiation (Fig. 4B). This observation was confirmed using a different EGFP-human Polκ construct (22Ogi T. Kannouche P. Lehmann A.R. J. Cell Sci. 2005; 118: 129-136Crossref PubMed Scopus (67) Google Scholar). Furthermore, foci were not detected (with or without UV radiation exposure) in cells transfected with EGFP-mouse Polκ lacking the UBZs (Fig. 4A). Similar results were obtained with EGFP-mouse Polκ carrying mutations in the UBZs (Fig. 4B). Hence, the UBZ domains are required for association of Polκ with replication factories in cells exposed to UV radiation. Mutation of the Polκ UBZs Does Not Alter the Half-life of the Protein–Given that Polκ is intrinsically error prone, regulation of Polκ levels is presumably important for maintenance of genetic integrity. To investigate the stability of Polκ in vivo, MEFs were treated with CHX for various lengths of time. Endogenous Polκ was degraded slowly with a half-life of 5.4 h (Fig. 5A). To determine whether the UBZ domains affect the half-life of the protein, COS7 cells were transfected with wild-type and UBZ mutant Polκ and were treated with CHX for various lengths of time. Although the turnover rate of these exogenous proteins (∼3.7–4.2 h) was relatively faster than that of endogenous Polκ, we observed essentially similar half-lives between wild-type and UBZ mutant Polκ (Fig. 5, B and C). Levels of Polκ Expression Are Increased in Cells Exposed to BPDE or UVB Radiation–Cells from two groups of independently generated Polκ knock-out mice are abnormally sensitive to BPDE and less so to UV radiation exposure (16Ogi T. Shinkai Y. Tanaka K. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15548-15553Crossref PubMed Scopus (200) Google Scholar, 17Schenten D. Gerlach V.L. Guo C. Velasco-Miguel S. Hladik C.L. White C.L. Friedberg E.C. Rajewsky K. Esposito G. Eur. J. Immunol. 2002; 32: 3152-3160Crossref PubMed Scopus (126) Google Scholar). To elucidate the underlying mechanism of this sensitivity, we examined the levels of nuclear Polκ after UVB and BPDE treatments. Examination of MEFs exposed to UVB radiation at different times revealed a progressive increase in the amount of Polκ 24–48 h after UVB exposure (Fig. 6A). Similarly, increased steady-state levels of Polκ were observed 8–30 h after exposure of MEFs to 1 mm BPDE for 1 h (Fig. 6B). To further support this conclusion, whole cell lysates were harvested at different times after exposure of MEFs to 1 mm BPDE for 1 h. Equal amounts of whole cell lysate were immunoprecipitated with rabbit anti-Polκ antibodies, and bound endogenous Polκ was detected with hamster anti-Polκ antibodies. Consistent with the results shown in Fig. 6B, increased levels of Polκ were observed 8–24 h after BPDE treatment (Fig. 6C). Persistent arrested DNA replication can threaten the viability of dividing cells. The observation that many eukaryotic cells, in particular those from higher eukaryotes, are endowed with multiple low fidelity specialized DNA polymerases that can catalyze DNA synthesis past sites of base damage in vitro has yielded important insights about DNA damage tolerance (2Friedberg E.C. Lehmann A.R. Fuchs R.P. Mol. Cell. 2005; 18: 499-505Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Regardless of the specific types of base damage in DNA handled by TLS, a question of considerable interest is how switching is effected at sites of arrested replication between high fidelity polymerases in the replicative machinery and one or more specialized enzymes that support TLS. Recent observations indicate that PCNA provides the central scaffold to which various TLS polymerases can bind to access the replicative ensemble stalled at a lesion and to execute their roles in lesion bypass (32Moldovan G.L. Pfander B. Jentsch S. Cell. 2007; 129: 665-679Abstract Full Text Full Text PDF PubMed Scopus (1346) Google Scholar, 33Huang T.T. D'Andrea A.D. Nat. Rev. Mol. Cell Biol. 2006; 7: 323-334Crossref PubMed Scopus (222) Google Scholar). However, it remains to be determined how a particular polymerase is selected to carry out TLS past a blocking lesion. To further our understanding of the biological role of Polκ during TLS in mammalian cells, we searched for interacting partners and identified ubiquitin. Hence, like the other Y-family polymerases, Polη, Polι, and Rev1 (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar, 26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar, 30Plosky B.S. Vidal A.E. Fernandez de Henestrosa A.R. McLenigan M.P. McDonald J.P. Mead S. Woodgate R. EMBO J. 2006; 25: 2847-2855Crossref PubMed Scopus (176) Google Scholar), Polκ binds ubiquitin. Although the precise biological function of this interaction remains to be determined, this binding likely reflects an interaction of Polκ with monoubiquitinated PCNA (31Bi X. Barkley L.R. Slater D.M. Tateishi S. Yamaizumi M. Ohmori H. Vaziri C. Mol. Cell. Biol. 2006; 26: 3527-3540Crossref PubMed Scopus (145) Google Scholar). The present study demonstrates that recently identified ubiquitin-binding motifs in Polκ (UBZs) are required for its interaction with PCNA, suggesting specific molecular events associated with the Polκ/PCNA interaction, especially in cells exposed to DNA-damaging agents such as UV radiation. Our studies represent the first demonstration of this phenomenon in living cells. Similar to the UBZs in Polη and the ubiquitin-binding motif in Polι (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar), the UBZs in Polκ are critical for the accumulation of the protein in replication foci when cells suffer DNA damage. Unlike the ubiquitin-binding motifs in REV1 (26Guo C. Tang T.S. Bienko M. Parker J.L. Bielen A.B. Sonoda E. Takeda S. Ulrich H.D. Dikic I. Friedberg E.C. Mol. Cell. Biol. 2006; 26: 8892-8900Crossref PubMed Scopus (169) Google Scholar), deletion/mutation of the UBZs completely abolished the basal level of focus formation by wild-type Polκ protein, suggesting that the basal level of Polκ foci may represent a response to spontaneous DNA damage. Surprisingly, the number of cells with visible mouse Polκ foci (∼55.3%) is significantly greater than that observed with human Polκ (∼25%) upon exposure to UV radiation (22Ogi T. Kannouche P. Lehmann A.R. J. Cell Sci. 2005; 118: 129-136Crossref PubMed Scopus (67) Google Scholar). The present studies also demonstrate that like other Y-family polymerases, mouse Polκ protein can be monoubiquitinated and that the UBZs in the protein are required for this modification. The biological significance of monoubiquitination of Y-family polymerases is not understood. However, this post-translational modification may contribute to regulation of Y-family polymerases in or out of replication factories (25Bienko M. Green C.M. Crosetto N. Rudolf F. Zapart G. Coull B. Kannouche P. Wider G. Peter M. Lehmann A.R. Hofmann K. Dikic I. Science. 2005; 310: 1821-1824Crossref PubMed Scopus (586) Google Scholar, 33Huang T.T. D'Andrea A.D. Nat. Rev. Mol. Cell Biol. 2006; 7: 323-334Crossref PubMed Scopus (222) Google Scholar). We reported previously the presence of multiple PolK transcripts in mouse testis (27Guo C. Gao T. Confer N. Velasco-Miguel S. Friedberg E.C. DNA Repair. 2005; 4: 397-402Crossref PubMed Scopus (11) Google Scholar). Many of the putative Polκ protein isoforms thus identified lack UBZ domains. It is thus of considerable interest to determine whether the putative Polκ isoforms are indeed expressed in vivo and what novel biological functions they may have. In addition to protein-protein interactions, Polκ activity may be regulated by its cellular levels. Polκ is apparently a relatively stable protein in vivo, and mutation of the UBZs does not alter the half-life of the protein. Consistent with this observation, the majority of Polκ in vivo is not monoubiquitinated, and polyubiquitinated Polκ is apparently absent. Polκ-deficient mouse and chicken cells manifest sensitivity to killing by BPDE (16Ogi T. Shinkai Y. Tanaka K. Ohmori H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15548-15553Crossref PubMed Scopus (200) Google Scholar), suggesting a specific requirement for Polκ to bypass this planar polycyclic lesion in DNA. Conceivably, adducts in DNA with similar planer polycyclic structures generated by cholesterol and cholesterol derivatives, such as steroid hormones and estrogen, generate the same requirement. Consistent with this notion, PolK mRNA is highly expressed in the adrenal cortex early during mouse embryonic development (28Velasco-Miguel S. Richardson J.A. Gerlach V.L. Lai W.C. Gao T. Russell L.D. Hladik C.L. White C.L. Friedberg E.C. DNA Repair. 2003; 2: 91-106Crossref PubMed Scopus (72) Google Scholar). We and other laboratories reported previously that the mouse PolK gene is transcriptionally up-regulated following exposure to UVB and BPDE treatments (28Velasco-Miguel S. Richardson J.A. Gerlach V.L. Lai W.C. Gao T. Russell L.D. Hladik C.L. White C.L. Friedberg E.C. DNA Repair. 2003; 2: 91-106Crossref PubMed Scopus (72) Google Scholar, 34Ogi T. Mimura J. Hikida M. Fujimoto H. Fujii-Kuriyama Y. Ohmori H. Genes Cells. 2001; 6: 943-953Crossref PubMed Scopus (87) Google Scholar), suggesting that exposure to these DNA-damaging agents promotes up-regulation of the gene. Consistent with this interpretation, the present studies demonstrate a progressive increase in steady-state levels of Polκ protein after such treatments. The biological significance of these expression patterns remains to be established. We thank Dr. Alan R. Lehmann for MRC5 cells and EGFP-hPolκ plasmid, Dr. Valerie Gerlach for preparing EGFP-C1-hPolκ construct and rabbit anti-Polκ, Drs. Tom Gillete and Lisa McDaniel for helpful discussion, and J. Nicole Kosarek for critical reading of the manuscript.
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