Requirements for the Interaction of Mouse Polκ with Ubiquitin and Its Biological Significance*

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) 3 is one of several biochemical mechanisms by which cells can tolerate DNA damage that arrests semiconservative DNA synthesis (1,2). 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.
Several specialized DNA polymerases are members of a novel polymerase family, the Y-family (3). These enzymes are devoid of 3Ј 3 5Ј proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processiv-ity (4). Members of this family in mammalian cells include Pol, Pol, and Pol, all of which can extend primers for varying distances past various types of template damage (4). 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 (5). Pol, Pol, and Pol have been shown to interact with REV1 protein via a highly conserved C-terminal domain in REV1 (6 -8). These polymerases also interact with PCNA (9,10), and recent observations suggest that PCNA plays a key role in promoting the access of specialized polymerases to arrested replication forks (11)(12)(13)(14)(15).
Disruption of the PolK gene in mouse and chicken cells results in significant sensitivity to killing by benzo[a]pyrenedihydrodiol epoxide (BPDE) and UV radiation (16 -18). Poldeficient mouse embryonic stem and fibroblast cells also show moderate sensitivity to methyl methanesulfonate (19). Consistent with these results, primer extension assays have shown that human Pol can support TLS across sites of base loss, acetylaminofluorene-G adducts, benzo[a]pyrene-G adducts, and thymine glycol (4). However, the enzyme does not support primer extension past thymine-thymine (TϽϾT) dimers or [6,4]pyrimidine-pyrimidone photoproducts (4). Similar to DNA polymerase Pol, Pol is also efficient in extending sites of replicative bypass by other specialized polymerases during TLS, at least in vitro (12). Furthermore, overexpression of Pol in mammalian cells promotes pleiotropic genetic alterations and tumorigenesis (20,21).
The relaxed fidelity of Pol 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 Pol accumulates in microscopically discrete nuclear foci in UV radiation-or BPDEtreated cells (22)(23)(24). In addition, the C-terminal 97 amino acids of Pol, which include a C2HC zinc finger, a bipartite nuclear localization signal, and a putative PCNA binding site, are important for the localization of Pol in nuclear foci (22). However, the underlying mechanism of TLS by Pol and other specialized DNA polymerases remains unclear.
To further our understanding of the role of Pol in TLS and in spontaneous and DNA damage-associated mutagenesis, we searched for proteins that interact with mouse Pol by screening a mouse testis cDNA library using the yeast two-hybrid system (6). We report here that ubiquitin binds strongly to Pol bait protein. We examined the interaction of ubiquitin with Pol and show that this interaction requires two novel zinc fingers, called ubiquitin-binding motifs (UBZs) (25), resident in * This study was supported in part by Grants ES11344 from NIEHS, National Institutes of Health (to E. C. F.), and DI 931/1-1 from the Deutsche Forschungsgemeinschaft (to I. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

EXPERIMENTAL PROCEDURES
Plasmids-For yeast two-hybrid screening, pGBT9/mouse PolK plasmid was cloned as described (6). 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.
Yeast Two-hybrid Assay-The pGBT9/mouse PolK plasmid was used to screen a mouse testis cDNA library as described (6).
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 (10).
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 (17). 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/m 2 was performed in a UV cross-linker (UV Stratalinker 2400, Stratagene). Nuclear extracts were harvested as described previously (27) at different time points.
Protein Half-life Determination-COS7 cells were transfected with the wild-type and UBZs mutant HA-Pol constructs. Twenty-four h later, the transfected cells were aliquoted into 8 -10 35-mm dishes to continue to culture for ϳ16 h. The half-life of Pol was determined by treating cells with 25 g/ml cycloheximide (CHX) (Sigma) for 0 -7 h to inhibit pro-tein synthesis and then preparing cell lysates to determine Pol levels by Western blotting. For endogenous Pol, wild-type MEFs were treated with 25 g/ml CHX for 0 -9 h. The content of Pol and ␤-actin bands was quantified by Photoshop histogram. To calculate the half-life of Pol protein, the content of the Pol Western blot bands at each time point was normalized to the control (0 h) Pol content and also refers to the ␤-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 Pol was made by our laboratory (27). Rabbit polyclonal antiserum against mouse Pol was generated with a 14-amino acid peptide (CNYLKIDTPRQE-ANE) containing an N-terminal cysteine residue conjugated with keyhole limpet hemocyanin as described (28). Anti-PCNA antibodies were purchased from Santa Cruz Biotechnology.
Lysate Preparation, Co-immunoprecipitation, and Western Blotting-COS7 cells were transfected with pCMV-HA-mPol and pCMV5-FLAG-Ub. Harvested cell lysates were immunoprecipitated with anti-FLAG antibodies. HEK293T cells were transfected with pCMV-Myc-mPol and pcDNA3-HA-Ub. Harvested cell lysates were immunoprecipitated with anti-Myc antibodies. Immunoprecipitation and immunoblotting were performed as described (26). MRC cells were transfected with HA-mPol, and 40 h later they were UV-irradiated (25 J/m 2 ). 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 (10).
GST Pulldown Assay-GST fusion proteins were expressed and purified on glutathione-agarose (Sigma) as described (6). Purified mPol was pulled down by GST-Ub as described previously (26). For interaction between truncated/mutant mPol 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 (10,26). Samples were separated by SDS-PAGE and detected by immunoblotting with polyclonal antibodies against mPol or with monoclonal antibodies against Myc (9E10), HA (16B12), or PCNA.
Immunofluorescence Microscopy-MRC5 cells were transfected using a panel of mutated/truncated EGFP-mPol and EGFP-hPol constructs and cultured for ϳ40 h. They were then UV-irradiated and processed for immunofluorescence as described previously (10). 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 Pol Interacts with Ubiquitin via Two Ubiquitinbinding Zinc Finger Domains and Undergoes Ubiquitination in
Vivo-By screening a mouse testis cDNA library using mouse Pol as bait, we identified both ubiquitin B and REV1 as inter-acting moieties. Because ubiquitin B is a polypeptide containing four tandem ubiquitin moieties, we anticipated that Pol would also bind to monoubiquitin. This was confirmed by GST pulldown using purified Pol and GST-ubiquitin (Fig. 1A). We observed that mouse Pol protein, in addition to binding ubiquitin, undergoes monoubiquitination in vivo. HA-Pol and FLAG-ubiquitin were expressed in COS7 cells, and cell lysates were immunoprecipitated with anti-FLAG antibodies. Not surprisingly, HA-Pol 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-Pol protein (Fig. 1B). This was directly confirmed by immunoprecipitation of Pol from cells cotransfected with Myc-Pol and HA-Ub followed by immunoblotting with anti-HA antibodies (Fig. 1C).
Sequence analysis revealed that the duplicated C2HC zinc cluster domains in mouse Pol (29) are in fact novel ubiquitinbinding domains called UBZs (25). The mouse UBZs (each ϳ30 amino acids in length) are located between amino acid residues 608 and 800 ( Fig. 2A). To determine whether the UBZs in Pol are required for binding ubiquitin, we incubated a fragment of Pol bearing just the two UBZs (UBZ1 ϩ UBZ2) with GSTubiquitin and confirmed the interaction (Fig. 2B). To further document the requirement of the UBZs in Pol for its interaction with ubiquitin, we generated a series of mutant constructs that deleted the N-terminal UBZ (UBZ1) (Pol-UBZ1⌬), the C-terminal UBZ (UBZ2) (Pol-UBZ2⌬), or both (Pol-UBZ⌬). 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 Pol UBZs were mutated to alanine (Fig. 2D). Mutational inactivation of the UBZs in Pol also impaired its monoubiquitination (Fig. 2E), and their deletion completely abolished monoubiquitination of Pol (Fig.  2E). Collectively, these results suggest that the Polk UBZs are required for interaction between Pol and ubiquitin and for monoubiquitination of the polymerase.
The UBZs Are Required for Enhanced Association between Pol 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 Pol, Pol, and REV1 protein (10,11,25,30). To determine whether an enhanced association also exists between Pol and monoubiquitinated PCNA, we examined their interaction by GST pulldown experiments (26). Consistent with results shown previously (31), the interaction of purified Pol 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 Pol 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-Pol 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 UBZdeleted, Pol was significantly increased after UVC treatment (Fig. 3C). Interestingly, the level of UBZ-deleted Pol 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 Pol was significantly increased after UVC treatment (Fig. 3D).
In summary, the results of the experiments reported thus far indicate that mouse Pol can interact with ubiquitin in vitro, an interaction that requires functional UBZs, and that Pol can itself undergo monoubiquitination. A robust association between monoubiquitinated PCNA and Pol also requires functional UBZs. Pol UBZs Are Required for Association of Pol with Replication Factories in Cells Exposed to UV Radiation-To validate the results described above in living cells, we transfected wildtype and UBZ-deleted EGFP-mouse Pol constructs into fibro-blasts. We observed strict nuclear localization of EGFP-mouse Pol protein, regardless of the presence or absence of the UBZs (Fig. 4A). As reported previously for human Pol (22), in ϳ4% of cells transfected with wild-type EGFP-mouse Pol the protein was concentrated in nuclear foci (Fig. 4A). When cells transfected with EGFP-mouse Pol were exposed to UV radiation and incubated for 8 -16 h, the fraction of cells with discrete nuclear foci increased to ϳ55.3% (Fig. 4B). Interestingly, the number of cells with mouse Pol 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 Pol construct (22). Furthermore, foci were not detected (with or without UV radiation exposure) in cells transfected with EGFP-mouse Pol lacking the UBZs (Fig. 4A). Similar results were obtained with EGFPmouse Pol carrying mutations in the UBZs (Fig. 4B). Hence, the UBZ domains are required for association of Pol with replication factories in cells exposed to UV radiation.
Mutation of the Pol UBZs Does Not Alter the Half-life of the Protein-Given that Pol is intrinsically error prone, regulation of Pol levels is presumably important for maintenance of genetic integrity. To investigate the stability of Pol in vivo, MEFs were treated with CHX for various lengths of time. Endogenous Pol 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 Pol and were treated with CHX for various lengths of time. Although the turnover rate of these exogenous proteins (ϳ3.7-4.2 h) was relatively faster than that of endogenous Pol, we observed essentially similar half-lives between wild-type and UBZ mutant Pol (Fig. 5, B and C).
Levels of Pol Expression Are Increased in Cells Exposed to BPDE or UVB Radiation-Cells from two groups of independently generated Pol knock-out mice are abnormally sensitive  to BPDE and less so to UV radiation exposure (16,17). To elucidate the underlying mechanism of this sensitivity, we examined the levels of nuclear Pol after UVB and BPDE treatments. Examination of MEFs exposed to UVB radiation at different times revealed a progressive increase in the amount of Pol 24 -48 h after UVB exposure (Fig. 6A). Similarly, increased steady-state levels of Pol 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-Pol antibodies, and bound endogenous Pol was detected with hamster anti-Pol antibodies. Consistent with the results shown in Fig. 6B, increased levels of Pol were observed 8 -24 h after BPDE treatment (Fig. 6C).

DISCUSSION
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 (2).
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 (32,33). 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 Pol during TLS in mammalian cells, we searched for interacting partners and identified ubiquitin. Hence, like the other Y-family polymerases, Pol, Pol, and Rev1 (25,26,30), Pol binds ubiquitin. Although the precise biological function of this interaction remains to be determined, this binding likely reflects an interaction of Pol with monoubiquitinated PCNA (31). The present study demonstrates that recently identified ubiquitinbinding motifs in Pol (UBZs) are required for its interaction with PCNA, suggesting specific molecular events associated with the Pol/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 Pol and the ubiquitin-binding motif in Pol (25), the UBZs in Pol are critical for the accumulation of the protein in replication foci when cells suffer DNA damage. Unlike the ubiquitin-binding motifs in REV1 (26), deletion/mutation of the UBZs completely abolished the basal level of focus formation by wild-type Pol protein, suggesting that the basal level of Pol foci may represent a response to spontaneous DNA damage. Surprisingly, the number of cells with visible mouse Pol foci (ϳ55.3%) is significantly greater  than that observed with human Pol (ϳ25%) upon exposure to UV radiation (22).
The present studies also demonstrate that like other Y-family polymerases, mouse Pol 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 (25,33).
We reported previously the presence of multiple PolK transcripts in mouse testis (27). Many of the putative Pol protein isoforms thus identified lack UBZ domains. It is thus of considerable interest to determine whether the putative Pol isoforms are indeed expressed in vivo and what novel biological functions they may have.
In addition to protein-protein interactions, Pol activity may be regulated by its cellular levels. Pol 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 Pol in vivo is not monoubiquitinated, and polyubiquitinated Pol is apparently absent.
Pol-deficient mouse and chicken cells manifest sensitivity to killing by BPDE (16), suggesting a specific requirement for Pol 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 (28).
We and other laboratories reported previously that the mouse PolK gene is transcriptionally up-regulated following exposure to UVB and BPDE treatments (28,34), suggesting that exposure to these DNA-damaging agents promotes upregulation of the gene. Consistent with this interpretation, the present studies demonstrate a progressive increase in steady-state levels of Pol protein after such treatments. The biological significance of these expression patterns remains to be established.