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• W2024703739 abstract "Article15 December 2003free access Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis Caixia Guo Caixia Guo Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Paula L. Fischhaber Paula L. Fischhaber Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Margaret J. Luk-Paszyc Margaret J. Luk-Paszyc Department of Physiology and Biophysics, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA Search for more papers by this author Yuji Masuda Yuji Masuda Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553 Japan Search for more papers by this author Jing Zhou Jing Zhou Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Kenji Kamiya Kenji Kamiya Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553 Japan Search for more papers by this author Caroline Kisker Caroline Kisker Department of Pharmacological Sciences, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA Search for more papers by this author Errol C. Friedberg Corresponding Author Errol C. Friedberg Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Caixia Guo Caixia Guo Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Paula L. Fischhaber Paula L. Fischhaber Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Margaret J. Luk-Paszyc Margaret J. Luk-Paszyc Department of Physiology and Biophysics, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA Search for more papers by this author Yuji Masuda Yuji Masuda Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553 Japan Search for more papers by this author Jing Zhou Jing Zhou Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Kenji Kamiya Kenji Kamiya Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553 Japan Search for more papers by this author Caroline Kisker Caroline Kisker Department of Pharmacological Sciences, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA Search for more papers by this author Errol C. Friedberg Corresponding Author Errol C. Friedberg Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA Search for more papers by this author Author Information Caixia Guo1, Paula L. Fischhaber1, Margaret J. Luk-Paszyc2, Yuji Masuda3, Jing Zhou1, Kenji Kamiya3, Caroline Kisker4 and Errol C. Friedberg 1 1Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9072 USA 2Department of Physiology and Biophysics, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA 3Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 734-8553 Japan 4Department of Pharmacological Sciences, Center for Structural Biology, State University of New York, Stony Brook, NY, 11794-5115 USA ‡C.Guo and P.L.Fischhaber contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6621-6630https://doi.org/10.1093/emboj/cdg626 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polκ and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Polκ. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Polζ) and with two other Y-family polymerases, Polι and Polη. Mouse Polκ, Rev7, Polι and Polη each bind to the same ∼100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Polκ for binding to the Rev1 C-terminus. Notwith standing the physical interaction between Rev1 and Polκ, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein–protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined. Introduction Recent years have witnessed the discovery of multiple specialized DNA polymerases in prokaryotic and eukaryotic cells (Friedberg et al., 2002; Goodman, 2002). Most of these enzymes belong to an evolutionarily related protein superfamily, the polymerase Y family (Ohmori et al., 2001), members of which are devoid of 3′→5′ proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity. The Y family of polymerases can replicate past a spectrum of template DNA damage by a process known as translesion synthesis (TLS). These features are shared by several other specialized polymerases from the A, B and X families. We and others previously reported features of the mouse and human PolK (DinB)/POLK (DINB) genes and their polypeptide products, DNA polymerase κ (Polκ) (Gerlach et al., 1999; Ohashi et al., 2000; Zhang et al., 2000). Primer extension assays have shown that human Polκ can support TLS across sites of base loss, acetylaminofluorene-G adducts, benzo[a]pyrene-G adducts (Hubscher et al., 2002) and thymine glycol (Fischhaber et al., 2002). However, the enzyme does not support primer extension past thymine–thymine (T<>T) dimers or [6-4] pyrimidine–pyrimidone photoproducts (Hubscher et al., 2002). Similar to another TLS DNA polymerase, Polζ, Polκ can extend terminal mismatches on undamaged templates in vitro (Haracska et al., 2002a; Prakash and Prakash, 2002; Washington et al., 2002). In addition, Polκ can extend primer-terminal nucleotides inserted opposite damaged bases by other specialized DNA polymerases (Frank et al., 2001; Haracska et al., 2002a; Zhang et al., 2002). Rev1 is also a member of the Y family of polymerases (Ohmori et al., 2001). In contrast to its relatives, Rev1 has limited catalytic activity in vitro, which is mainly reflected in the preferential and limited incorporation of dCMP in a template-directed manner regardless of the template nucleotide (Nelson et al., 1996). Rev1 is required for error-prone TLS by polζ, but its dCMP transferase activity is not obligatory for this function (Baynton et al., 1999; Nelson et al., 2000; Lawrence, 2002). Rev1 is unable to support TLS across pyrimidine dimers or [6-4] photoproducts. Nonetheless, the REV1 gene is required for UV radiation-induced mutagenesis in yeast and human cells (Lawrence, 2002). Collectively, these observations suggest that Rev1 plays an as yet unidentified role(s) in TLS that is unrelated to the dCMP transferase activity. This suggestion is supported by recent studies showing that chicken DT40 cells in which the nucleotidyl transferase domain and C-terminal domain of Rev1 protein have been inactivated are abnormally sensitive to a variety of DNA-damaging agents (Simpson and Sale, 2003). To further our understanding of the role of Polκ in TLS and mutagenesis, we have searched for proteins that interact with mouse Polκ (mPolκ). Here we show that mPolκ specifically interacts with mouse Rev1 protein (mRev1). We have mapped a limited C-terminal domain of mRev1 that is necessary and sufficient for this interaction. Importantly, we observed that mRev1 interacts with several other specialized DNA polymerases, notably mPolι, mPolη and the Rev7 subunit of the heterodimeric specialized polymerase mPolζ. In each case, the limited C-terminal domain of mRev1 is required for these interactions. We also show that the catalytic activities of mRev1 and mPolκ acting in concert are not detectably altered when copying undamaged, normally base-paired DNA in vitro. However, in particular template DNA sequence contexts, mRev1 and mPolκ together exhibit primer extension activity that is greater than the additive activity of the individual polymerases when synthesizing DNA past a thymine glycol base or extending certain mismatched primer-termini. We show that this enhanced activity does not derive from Polκ/Rev1 protein complex formation. Results Mouse Polκ interacts with mouse Rev1 protein To identify proteins that interact with mPolκ, a mouse testis cDNA library constructed in the two-hybrid vector pACT2 was screened using mPolκ (amino acids 100–616) as bait. Prior to the screen we determined that the bait alone did not yield transactivation in the assay. We screened ∼6 × 106 clones on quadruple drop-out (QDO) plates depleted for adenine (ade), histidine (his), leucine (leu) and tryptophan (trp). Four resulting positive colonies represented fragments of the Y-family protein mRev1 (amino acids 239–1249, 632–1249, 767–1249 and 871–1249). Yeast containing both mPolκ- and Rev1-expressing plasmids were able to grow on QDO plates (Figure 1A), whereas if either of the plasmids contained no insert, there was no growth on QDO plates (Figure 1A). Interaction between mPolκ and mRev1 in the yeast two-hybrid system was further confirmed by measuring β-galactosidase activity from a lacZ reporter gene in extracts of cells transformed with relevant plasmid pairs (Figure 1B). Figure 1.Interaction between mPolκ and mRev1. (A) AH109 was co-transformed with plasmid combinations as indicated and plated on QDO medium. The combinations tested were: 1, mDinB-pGBT9 + Rev1-pGADT7; 2, mDinB-pGBT9 + pGADT7; 3, mRev1-pGADT7 + pGBT9; 4, pGBT9 + pGADT7. Only the mDinB-pGBT9 + Rev1-pGADT7 combination was viable. The presence of ‘bait’ and ‘prey’ plasmids in co-transformed cells was controlled by growth on DDO media. (B) Extracts prepared from yeast transformed with plasmid combinations described above were assayed for β-galactosidase activity. Values are in Miller units. Data represent the average of three independent experiments with error bars representing standard deviations. (C) Association between mouse Polκ and Rev1 in cos7 cells. Lysates from HA-mPolκ and Myc-mRev1 co-transfected cos7 cells were analyzed by immunoprecipitation and western blotting, as indicated. A mock antibody (normal rabbit serum) was used in controls. Input lanes contained 1/25 the lysates used in the experiments. Top panel, Myc-mRev1 co-immunoprecipitates with HA-mPolκ. Bottom panel, HA-mPolκ co-immunoprecipitates with Myc-mRev1. (D) Immuno precipitation with a mixture of 0.3 μM each of purified mRev1 and mPolκ. Upper panel, the blot was probed with anti-mPolκ antibody. Lane 1 contains 1/35 the amount of purified mPolκ used in the reactions. Lanes 2–5 show immunoprecipitation of the mRev1/mPolκ mixture with the following: lane 2, normal rabbit serum; lane 3, anti-Rev1 serum with mRev1 omitted; lane 4, anti-Rev1 serum with mPolκ omitted; lane 5, anti-Rev1 serum. Lower panel, the blot was stripped and probed with anti-Rev1 antibody. IP and IB indicate immunoprecipitate and immunoblot, respectively. Download figure Download PowerPoint Interaction between mRev1 and mPolκ was also demonstrated by immunoprecipitation. Mouse Polκ and mouse Rev1 proteins tagged with HA (HA-mPolκ) and Myc (Myc-mRev1) epitopes at their N-termini were expressed from mammalian expression vectors. Western analysis using antibodies specific to the HA or Myc epitopes confirmed co-expression in cos7 cells (Figure 1C). Cell lysates were immunoprecipitated with either anti-HA or anti-Myc polyclonal antibodies using normal rabbit serum as a mock control (Figure 1C). HA-mPolκ co-precipitated with Myc-mRev1 regardless of which antibody was used for immunoprecipitation or western analysis. However, neither protein was detected when rabbit serum was used as an immunoprecipitation control (Figure 1C). A mixture of stoichiometric equivalents of purified mRev1 and mPolκ was also co-precipitated with anti-Rev1 antibody (Figure 1D, lane 5), while anti-Rev1 antibody did not precipitate mPolκ alone (Figure 1D, lane 3). Interaction between mPolκ and mRev1 requires the C-terminal 100 amino acids of mRev1 The shortest of the four polypeptides that interacted with mPolκ (amino acids 100–616) in the two-hybrid screen contains the C-terminal 378 amino acids, suggesting that a limited C-terminal region of mRev1 is necessary and sufficient for interaction with mPolκ. To map this region more precisely we constructed mRev1 cDNAs carrying various deletions and tested these in two-hybrid assays. Clones containing the C-terminal 100 amino acid residues (amino acids 1150–1249) yielded positive interactions, whereas clones expressing smaller mRev1 polypeptides did not (Figure 2A). Figure 2.Deletion mapping of mRev1 region required for interaction with mPolκ (A and B). (A) Deletion mutants of mRev1 were tested for their ability to interact with full-length mPolκ in the yeast two-hybrid system. On auxotroph selective plates (QDO + x-α-gal), yeast co-transformed with full-length or truncated mRev1 constructs 1–5 plus the mDinB plasmid are viable, showing blue colonies within 3 days. In contrast, yeast co-transformed with truncated mRev1 constructs 6–7 plus the mPolκ plasmid are not viable. (B) In vitro-translated Myc-mPolκ was added to glutathione beads coupled with either GST (lane 2), GST–Rev1-3 (lane 3), GST–Rev1-4 (lane 4), GST–Rev1-5 (lane 5) or GST–Rev1-6 (lane 6) fusion proteins. The input lane (lane 1) contains 1/20 of the IVTT product used in the experiment. Interactions were examined by western analysis using monoclonal antibody against Myc. (C) Deletion mapping of the mPolκ region required for interaction with mRev1. Deletion mutants of mPolκ were tested for their ability to interact with full-length mRev1 in the yeast two-hybrid system as described above. Download figure Download PowerPoint To confirm these results, mPolκ was incubated with GST-tagged mRev1 proteins expressed from various deletion constructs and coupled to glutathione–agarose beads. The washed beads were resuspended in SDS loading buffer and bound proteins were detected by western analysis using monoclonal antibody against the Myc epitope. Once again mPolκ only bound GST–mRev1 polypeptides that included the C-terminal amino acids 1150–1249 (Figure 2B). The reciprocal experiment was performed in which proteins expressed from different deletion constructs of mouse PolK cDNA were examined for interaction with full-length mRev1 protein with the two-hybrid assay. Only truncated mPolκ polypeptides that included amino acid residues 230–616 (present in the bait protein for the initial two-hybrid screen) yielded interactions (Figure 2C). This region of mPolκ includes two conserved helix–hairpin–helix (HhH) domains as well as an undefined domain conserved in all members of the DinB subfamily of the Y superfamily (Gerlach et al., 1999). Mouse Rev1 interacts with other novel DNA polymerases through its C-terminal domain We examined the interaction of Rev1 with other specialized DNA polymerases known to support TLS in vitro or in vivo. Using the yeast two-hybrid system we observed interactions between mRev1 and mPolι (Figure 3A) and between mRev1 and mPolη (Figure 3B). To confirm these results we co-transfected Flag and Myc epitope-tagged constructs (Flag-mPolι and Myc-mRev1; Flag-mPolη and Myc-mRev1) into cos7 cells and incubated cell lysates with anti-Flag affinity beads. Precipitated proteins were detected by western analysis using antibodies against the Myc or Flag epitopes. Mouse Rev1 protein was shown to interact with mPolι (Figure 3C) and mPolη (Figure 3D). The specificity of these interactions was demonstrated with appropriate controls (Figure 3C and D). As observed with the mRev1/mPolκ interaction the C-terminal 100 amino acids of Rev1 are sufficient for interaction with mPolι or mPolη (Figure 3). Neither of these proteins interacted with mPolκ in yeast two-hybrid and immunoprecipitation experiments (data not shown). Figure 3.Deletion mapping of mRev1 to determine the minimal region required for interaction with mPolι (A) or mPolη (B) by the yeast two-hybrid assay. (C) Association between mRev1 and mPolι in cos7 cells. Anti-Flag M2 agarose affinity gel was incubated with the cos7 cell lysates expressing Myc-mRev1 and Flag-mPolι or Myc-mRev1 (control). Top panel, immunoblotting to detect Myc-mRev1. Lanes 1 and 2, input containing 1/50 the lysate used for immunoprecipitation. Lanes 3 and 5, immunoprecipitation of lysates with anti-Flag M2 antibody. The lysates express Myc-mRev1 (lane 3) or Myc-mRev1 and Flag-mPolι (lane 5), respectively. Lane 4, Myc-mRev1 and Flag-mPolι lysates were precipitated with mock antibody (HA). Bottom panel, the blot was stripped and probed with anti-Flag monoclonal antibody. (D) Interaction between mRev1 and mPolη in cos7 cells. Lysates expressing Myc-mRev1 and Flag-mPolη were precipitated and detected analogously to (C). Download figure Download PowerPoint It has been previously shown that human Rev1 protein interacts with human Rev7 protein, a subunit of the heterodimeric TLS polymerase Polζ, and that the Rev7-binding domain resides in the Rev1 C-terminus (amino acids 1130–1251) (Murakumo et al., 2001; Masuda et al., 2003). Since this region of Rev1 is highly conserved between the mouse and human proteins, we examined mRev1 and mRev7 proteins for this interaction. Cell lysates expressing Myc-mRev1 and Flag-mRev7 were precipitated with anti-Flag affinity beads. Western analysis of the precipitates using an anti-Myc monoclonal antibody demonstrated co-precipitation of mRev1 with Flag-mRev7 (Figure 4A). No mRev1 protein was detected in control experiments in which only the Myc-mRev1 lysate was precipitated (Figure 4A). This interaction was confirmed with in vitro GST pull-down experiments. Recombinant mRev1 was incubated with equal amounts of either GST or GST–mRev7 proteins and bound to glutathione–agarose beads. After extensive washes bound proteins were analyzed by western analysis using polyclonal antibody against human Rev1 (amino acids 245–847). GST–mRev7 protein bound specifically to mRev1 (Figure 4B, lane 4). Diminished amounts of mRev1 were recovered with the GST–mRev7 beads in the presence of increasing amounts of purified mPolκ (Figure 4B, lanes 5 and 6), suggesting that mRev7 and mPolκ compete directly for binding to mRev1. Mouse Polκ and mRev7 did not interact in the yeast two-hybrid system or in GST pull-down assays (data not shown). Figure 4.Association between mRev1 and mRev7. (A) Extracts of cos7 cells expressing Flag-mRev7 and Myc-mRev1 were incubated with anti-Flag M2 agarose affinity gel. Retained proteins were detected by immunoblotting with monoclonal antibody against Myc. Input lanes contain 1/40 of the lysates used in the experiments. (B) GST pull-down of mRev1 with GST–Rev7. Recombinant mRev1 (45 nM) was incubated with 40 μg GST or GST–Rev7 coupled to glutathione beads in the absence or presence of purified mPolκ. Bound proteins were resolved by 8% SDS–PAGE followed by immunoblot analysis with anti-Rev1 antibody. Lane 1 contains 1/10 of the mRev1 used in the experiments. Lane 2, GST+mRev1; lane 3, GST + mRev1 + 450 nM mPolκ; lane 4, GST–Rev7 + mRev1; lane 5, GST–Rev7 + mRev1 + 45 nM mPolκ; lane 6, GST–Rev7 + mRev1 + 450 nM mPolκ. (C) Rev7 competes with Polκ for binding to the mRev1 C-terminus. Immobilized GST–Rev1-4 (amino acids 1124–1249) fusion protein (5 μg) was incubated with a fixed amount of recombinant mPolκ (5 nM) in the presence of increasing concentrations (0–200 nM) of GST–Rev7 or GST, as indicated. Bound proteins were resolved by 8% SDS–PAGE followed by immunoblot analysis with anti-mPolκ antibody (top panel). The blot was stripped and probed with anti-Rev7 antibody (bottom panel). As more GST–Rev7 protein is added, there is an increase in GST–Rev7 binding and a decrease in mPolκ binding to GST–Rev1-4. Data are representative of three independent experiments. Download figure Download PowerPoint Since the C-terminal 100 amino acids of mRev1 protein are required for binding to both mPolκ and mRev7, and in light of the observation that mPolκ and mRev7 may compete directly for binding to mRev1, we examined interactions between the three proteins GST–Rev1-4 (amino acids 1124–1249), mPolκ and mRev7. The C-terminal region of mRev1 was bound to glutathione–agarose beads and washed extensively with GST protein until saturated. Bound beads were incubated with a fixed concentration of mPolκ followed by increasing amounts of GST–mRev7 protein eluted from glutathione beads. Following extensive washing the association between Polκ and GST–Rev1-4 was evaluated by immunoblot analysis with a polyclonal antibody against mPolκ. Increasing amounts of GST–mRev7 protein in the incubation (but not GST alone) decreased the amount of Polκ bound to GST–Rev1-4 and increased the amount of GST–Rev7 bound (Figure 4C). In summary, our results demonstrate that mouse Polκ, Polι, Polη and Rev7 each interact with mRev1. In the cases of mPolκ, mPolι and mPolη, the C-terminal 100 amino acids of mRev1 are sufficient for the interaction. Additionally, mPolκ and mRev7 compete directly for binding to mRev1 protein. Catalytic activity of the mouse Polκ/Rev1 complex In view of the observation that purified mPolκ and mRev1 form a stable complex in solution (Figure 1D) we compared the polymerase activity of each alone and when incubated together. The two proteins were introduced into reaction mixtures by mixing droplets of each on the side of incubation tubes and gently pushing the mixed droplets into reactions containing the remaining components pre-warmed to 37°C. When individual protein droplets were mixed for a minute prior to addition to the primer extension reaction no differences were noted compared to shorter mixing times, indicating that 1–2 s was sufficient for mPolκ/mRev1 to attain binding equilibrium. We also performed experiments in which each of the four dNTPs were introduced individually to demonstrate that the fidelity of mPolκ or mRev1 was unaltered when replicating normally base-paired DNA together. Experiments with a native and correctly base-paired primer-template. Visual examination and quantitation (shown below each lane in Figure 5A) of individual radiolabeled bands generated by primer extension of undamaged template DNA normally base-paired with a primer demonstrated that, in the presence of both mPolκ and mRev1, the relative quantities of total nucleotides incorporated were essentially the sum of that observed with each polymerase alone (Figure 5A). Additionally, replication fidelity was unaltered (data not shown). We conclude that neither DNA polymerase activity nor fidelity is significantly altered when a mixture of the two polymerases extends a normally base-paired primer annealed to undamaged template DNA. Figure 5.Direct interaction does not influence the polymerase activities of mRev1 and mPolκ in vitro on undamaged base-paired primer-templates or opposite a thymine glycol template base. Radiolabeled DNA primer-templates and the four dNTPs were incubated with mPolκ, mRev1 or both proteins. Reaction products were resolved by DPAGE. For each panel: lane 1, control with no enzyme; lanes 2, 3 and 4, mPolκ alone at 0.5, 1 and 5 nM, respectively; lanes 5, 6 and 7, mPolκ and mRev1 at 0.5, 1 and 5 nM each, respectively; lanes 8, 9 and 10, mRev1 alone at 0.5, 1 and 5 nM respectively. (A) Undamaged base-paired primer-template substrate (local sequence context indicated in the scheme above the gel). (B) Primer-template substrate containing a single thymine glycol base (local sequence context indicated in the scheme above the gel); thymine glycol is represented as ‘Tg’. (C and D) Analogous to (A) and (B) except that the nucleotidyl transferase-defective mRev1AA protein was used instead of wild-type mRev1. (E and F) Analogous to (A) and (B) except that the C-terminal deletion mRev1ΔC protein lacking the domain for binding to mPolκ was used instead of wild-type mRev1. The position on the gels corresponding to the primer extended by a single nucleotide (opposite template T or template Tg) is indicated by an arrow to the right of the gel. The total quantity of deoxynucleotides incorporated/reaction is indicated below each lane of each gel as pmol. Download figure Download PowerPoint Experiments with template DNA containing base damage. Human Polκ can efficiently bypass thymine glycol bases during primer extension in vitro (Fischhaber et al., 2002). In the present studies we asked whether mPolκ is also endowed with this property and, if so, whether this property is altered in the presence of mRev1. Experiments were performed using a DNA oligonucleotide containing thymine glycol as the next template base. The polymerase activity in each primer extension reaction was determined quantitatively and expressed as total deoxynucleotide incorporated per reaction (see results below each lane in Figure 5B). Like the human protein, mPolκ is able to support efficient TLS across thymine glycol (Figure 5B). Additionally, mRev1 bypasses thymine glycol (Figure 5B). In this case the polymerase activity when the two proteins were incubated together was greater than the additive activities of each alone (Figure 5B). For example, 1.9 pmol of dNMP was incorporated by mPolκ (Figure 5B, lane 4) and 0.3 pmol was incorporated by mRev1 (Figure 5B, lane 10). However, when the two enzymes were incubated together 4.6 pmol rather than the expected 2.2 pmol of dNMP was incorporated (Figure 5B, lane 7). This result suggests that physical interaction between mPolκ and mRev1 during primer extension stimulates nucleotide incorporation opposite thymine glycol. However, the two template bases immediately following thymine glycol are both G. Thus, an alternative explanation for the stimulation derived from known properties of Rev1 protein. Mouse Rev1 may realign the template by skipping the thymine glycol and incorporate C opposite the two template G residues. If the resulting partially extended primer-template provides a better substrate for mPolκ, more robust extension beyond the lesion may result, a scenario in accord with the two-step, two-polymerase model for TLS proposed by others (Bridges and Woodgate, 1985; Johnson et al., 2000; Pages and Fuchs, 2002). To distinguish between these two possibilities we generated mutant forms of mRev1 protein. In one case the nucleotidyl transferase domain of mRev1 was inactivated by changing conserved aspartate and glutamate residues to alanine (D568A, E569A, designated mRev1AA). This form of mRev1 is expected to retain a stimulatory effect promoted by protein–protein interaction, even though it is unable to support nucleotide incorporation. In the other case mRev1 was deleted of the C-terminal region required for interaction with mPolκ (designated mRev1ΔC). This form of mRev1 is expected to retain catalytic activity but to be inactive for stimulation mediated by interaction between the two proteins. Mouse Rev1AA protein was devoid of polymerase activity, while mRev1ΔC retained the ability to support incorporation of C (Figure 5C and E). However, the amount of primer extension with mPolκ and mRev1AA was identical to that observed with mPolκ alone, using either an undamaged template (Figure 5C) or the thymine glycol template (Figure 5D). Similarly the extent of primer extension with mPolκ and mRev1ΔC (Figure 5E and F) was identical to that supported by mPolκ and mRev1 (Figure 5A and B). Collectively, these results support the two-step, two-polymerase model for TLS and indicate that mixing mPolκ and mRev1 does not significantly alter the polymerase function of either polymerase when bypassing thymine glycol in vitro. Experiments with mispaired primer-termini. Polκ can extend mispaired primer termini (Washington et al., 2002). We examined such primer extension by a mixture of mPolκ and mRev1 proteins using annealed primers terminating either in a correct base pair (A:T) or a mismatched base pair (C:T). Once again the correctly paired A:T substrate showed little difference in primer extension when mPolκ was incubated alone or with mRev1 (Figure 6A). However, the C:T mismatched substrate supported slightly enhanced activity when incubated with mRev1 (Figure 6B). In experiments using the mRev1AA and mRev1ΔC mutant proteins the results were completely analogous to those obtained with thymine glycol (Figure 6C–F). We performed an additional primer extension experiment in whic" @default.
• W2024703739 created "2016-06-24" @default.
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• W2024703739 date "2003-12-15" @default.
• W2024703739 modified "2023-10-16" @default.
• W2024703739 title "Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis" @default.
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• W2024703739 doi "https://doi.org/10.1093/emboj/cdg626" @default.
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