Title: Mechanisms of dCMP Transferase Reactions Catalyzed by Mouse Rev1 Protein
Abstract: The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the Vmax value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at aVmax similar to that for dCMP. The Rev1 protein, a member of a large family of translesion DNA polymerases, catalyzes a dCMP transfer reaction. Recombinant mouse Rev1 protein was found to insert a dCMP residue opposite guanine, adenine, thymine, cytosine, uracil, and an apurinic/apyrimidinic site and to have weak ability for transfer to a mismatched terminus. The mismatch-extension ability was strongly enhanced by a guanine residue on the template near the mismatched terminus; this was not the case with an apurinic/apyrimidinic site and the other template nucleotides. Kinetic analysis of the dCMP transferase reaction provided evidence for high affinity for dCTP with template G but not the other templates, whereas the template nucleotide did not much affect the Vmax value. Furthermore, it could be established that the mouse Rev1 protein inserts dGMP and dTMP residues opposite template guanine at aVmax similar to that for dCMP. apurinic/apyrimidinic. BRCT, BRCA1 C terminus In yeast Saccharomyces cerevisiae, the REV1gene is required for damage-induced and spontaneous mutagenesis (1Glassner B.J. Rasmussen L.J. Najarian M.T. Posnick L.M. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9997-10002Crossref PubMed Scopus (176) Google Scholar, 2Kalinowski D.P. Larimer F.W. Plewa M.J. Mutat. Res. 1995; 331: 149-159Crossref PubMed Scopus (11) Google Scholar, 3Lawrence C.W. Christensen R. Genetics. 1976; 82: 207-232Crossref PubMed Google Scholar, 4Lawrence C.W. Christensen R.B. J. Mol. Biol. 1978; 122: 1-21Crossref PubMed Scopus (58) Google Scholar, 5Lawrence C.W. Stewart J.W. Sherman F. Christensen R. J. Mol. Biol. 1974; 85: 137-162Crossref PubMed Scopus (95) Google Scholar, 6Lawrence C.W. Stewart J.W. Sherman F. Thomas F.L.X. Genetics. 1970; 64: 836-837Google Scholar, 7Lemontt J.F. Genetics. 1971; 68: 21-23Crossref PubMed Google Scholar). A defect in the REV1 gene has in fact been found to decrease the translesion replication of apurinic/apyrimidinic (AP)1 sites, T-T (6-4) UV photoproducts, and N-2-acetylaminofluorene-modified guanine (8Baynton K. Bresson-Roy A. Fuchs R.P. Mol. Microbiol. 1999; 34: 124-133Crossref PubMed Scopus (58) Google Scholar, 9Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (184) Google Scholar). The encoded protein, containing a BRCA1 C terminus (BRCT) domain at its N terminus, possesses deoxycytidyl transferase activity (10Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (485) Google Scholar, 11Larimer F.W. Perry J.R. Hardigree A.A. J. Bacteriol. 1989; 171: 230-237Crossref PubMed Google Scholar, 12Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar) inserting dCMP residues opposite templates G, A, U, and AP sites (12Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar). The activity of Rev1 protein could be important for the bypass of AP sites in yeast (9Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (184) Google Scholar) because cytidine is preferentially inserted opposite these lesionsin vivo (9Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (184) Google Scholar, 13Gibbs P.E. Lawrence C.W. J. Mol. Biol. 1995; 251: 229-236Crossref PubMed Scopus (83) Google Scholar). However, a number of observations have suggested that the Rev1 protein may possesses a second function. First, when the REV1 gene is required for the bypass of a T-T (6-4) UV photoproduct, dCMP incorporation occurs only very rarelyin vivo (9Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (184) Google Scholar). Second, translesion DNA synthesis and mutagenesis are greatly reduced in a rev1 mutant,rev1–1, with a BRCT domain alteration that does not affect the deoxycytidyl transferase activity in vitro (9Nelson J.R. Gibbs P.E. Nowicka A.M. Hinkle D.C. Lawrence C.W. Mol. Microbiol. 2000; 37: 549-554Crossref PubMed Scopus (184) Google Scholar, 11Larimer F.W. Perry J.R. Hardigree A.A. J. Bacteriol. 1989; 171: 230-237Crossref PubMed Google Scholar). Third, during bypass of N-2-acetylaminofluorene-modified guanine, the REV1 gene is needed only for non-slipped translesion DNA synthesis, suggesting that the uncharacterized Rev1 activity is UmuDC-like in nature (8Baynton K. Bresson-Roy A. Fuchs R.P. Mol. Microbiol. 1999; 34: 124-133Crossref PubMed Scopus (58) Google Scholar). Fourth, methyl methanesulfonate-induced mutagenesis was shown to be normal in a site-directed mutant lacking deoxycytidyl transferase activity (14Haracska L. Unk I. Johnson R.E. Johansson E. Burgers P.M. Prakash S. Prakash L. Genes Dev. 2001; 15: 945-954Crossref PubMed Scopus (291) Google Scholar). Fifth, +1 frameshift mutations accompanying base substitutions are dependent on the REV1 gene (15Harfe B.D. Jinks-Robertson S. Mol. Cell. 2000; 6: 1491-1499Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Recently, cloning of a human homologue of the REV1 gene (16Gibbs P.E. Wang X.D. Li Z. McManus T.P. McGregor W.G. Lawrence C.W. Maher V.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4186-4191Crossref PubMed Scopus (162) Google Scholar,17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar) revealed good conservation from yeast to humans. The humanREV1 gene encodes a deoxycytidyl transferase, similar to the Rev1 protein of the yeast, S. cerevisiae (17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar, 18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), with activity localized to the central domain that is conserved in the UmuC superfamily (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Mutations in conserved residues but not the BRCT domain completely abolish the transferase activity (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Proteins in the UmuC superfamily, except for the Rev1 protein, are novel DNA polymerases, capable of replicating damaged DNA (19Ohmori 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). Only for the Rev1 protein has no polymerase activity been detected so far. It has been reported that its transferase activity is limited to the insertion of a dCMP residue (12Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar). Although the enzyme is capable of incorporating a dCMP residue not only opposite G but also A, U, and AP sites (12Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar), it is not clear how the Rev1 protein plays a role in mutagenesis. In the present study, we cloned and characterized the Rev1gene of the mouse, an animal commonly used for models of human disease. We found that the mouse Rev1 protein transfers a dCMP residue not only opposite template G but also A, T, C, U, and AP sites and extends a mismatched terminus by the addition of a dCMP residue. We further showed this mismatch-extension ability to be strongly enhanced by the presence of a guanine residue (but not an AP site) on the template near the mismatched terminus. Kinetic analysis of the dCMP transferase reaction provided evidence for the high affinity of dCTP with template G. Furthermore, the mouse Rev1 protein could be shown to insert dGMP and dTMP residues opposite template guanine and AP sites. The same activity was also detected with recombinant human REV1 protein but not an inactive mutant protein. Mice of the C57BL/6N and C3H/He strains were purchased from Charles River Laboratories Inc., Atsugi, Japan. All experiments followed the guidelines of the Animal Experimental Facility Committee of Hiroshima University. Poly(A)+ mRNA was isolated from the liver of an inbred mouse, strain C3H/He, using a poly(A) tract mRNA isolation system (Promega). Double-stranded cDNA was synthesized using a cDNA synthesis kit (Takara), and PCR primers corresponding to the human REV1 gene were used to amplify fragments of mouse Rev1 cDNA. The amplified fragments were cloned and sequenced. From the sequence information, several primer sets were designed and tested. Consequently, mouse Rev1 cDNA was successfully amplified as three overlapping cDNA fragments usingPyrobestTM DNA polymerase (Takara). A promoter proximal fragment (fragment I) was amplified with primers (5′-GAAGCTCCCATATGAGGCGA-3′ and 5′-CTGAAGTTGAGCTGTTTGGC-3′) at 55 °C for annealing with 30 cycles. A fragment of the central region (fragment II) was amplified with primers (5′-AATCCTGTGTGCAAACCTGAG-3′ and 5′-TTTCGTCTCTGCAAGGATGTC-3′), and a promoter distal fragment (fragment III) was amplified with primers (5′-TGCGATGAAGCACTGATTGAC-3′ and 5′-TCAGGTCACTTTCAGTGTGCT-3′) under the same conditions. These fragments were cloned into a pCR 2.1-TOPO vector (Invitrogen). Several independently isolated clones were sequenced, and mutant clones with different sequences from the others were rejected. A plasmid containing wild-type fragment I was digested with NdeI andBamHI, a plasmid containing fragment II was digested withBamHI and AatII, and a plasmid containing fragment III was digested with AatII and KpnI and assembled into the pCR 2.1-TOPO vector. To make a bacterial expression plasmid, the human REV1 fragment of pBADREV1 (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) was replaced with the mouse Rev1 fragment. The resulting plasmid, pBADmRev1, encodes full-length mouse Rev1 protein tagged with hexa-histidine residues at its N terminus (h6-mRev1). The tagged sequence is identical with that of pET15b (Novagen). Gene expression was induced by arabinose. The sequence data for the mouseRev1 cDNA have been submitted to the DDBJ/EMBL/GenBankTM data bases under accession numberAB057418. A Northern blot membrane with 2 μg of poly(A)+ mRNAs from tissues of C57BL/6N × C3H/He mice was hybridized with a 32P-labeled probe generated by PCR using primers 5′-TCCCAGATTGACCAGTCTGTT-3′ and 5′-TCAGGTCACTTTCAGTGTGCT-3′ in ExpressHybTM Hybridization Solution (CLONTECH) at 65 °C and washed with 0.1 × SSC and 0.1% SDS at the same temperature. Signals were visualized by autoradiography at −80 °C. BL21(DE3) harboring pBADmRev1 was grown in 500 ml of SB medium (20Mayer M.P. Gene (Amst.). 1995; 163: 41-46Crossref PubMed Scopus (185) Google Scholar) supplemented with ampicillin (100 mg/ml) at 15 °C with aeration until the culture reached an A600 value of 0.6. l(+)-arabinose was added to 1%, and the incubation was continued for 10 h. The cells were harvested, and cell lysate was prepared as described previously (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Subsequent column chromatography was carried out at 4 °C using a SMART system (Amersham Biosciences, Inc.). Two-ml aliquots of lysate were applied at 0.1 ml/min to a 1-ml HiTrap chelating column (Amersham Biosciences, Inc.), which had been flushed with 2 ml of 0.1 m NiSO4 and then equilibrated with buffer A (50 mm HEPES-NaOH, pH 7.5, 1 mNaCl, 10% glycerol, 10 mm β-mercaptoethanol) containing 10 mm imidazole, 5 mm ATP, and 10 mm MgCl2. The column was washed with 10 ml of equilibration buffer and then with 12 ml of buffer A containing 100 mm imidazole, 5 mm ATP, and 10 mmMgCl2. The h6-mRev1 was eluted with buffer A containing 300 mm imidazole, 5 mm ATP, and 10 mmMgCl2. Five hundred μl of the peak fraction of the h6-mRev1 was concentrated using an Ultrafree-0.5 centrifugal filter device, Biomax-10 (Millipore), and then applied at 0.01 ml/min to a Superdex 200 PC 3.2/30 column (Amersham Biosciences, Inc.), equilibrated with buffer A, and 40-μl fractions were collected. Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin (BSA) (Bio-Rad) as a standard. The recombinant human REV1 protein and its mutant, D569A/E570A, were purified by nickel-chelating column and gel filtration chromatography as described (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Sucrose density gradient sedimentation was performed as described previously (21Maki H. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4389-4392Crossref PubMed Scopus (94) Google Scholar). The purified h6-mRev1 protein (4 μg) was sedimented through 2 ml of 10–40% sucrose gradient in buffer A by centrifugation at 55,000 rpm for 15 h in a TLS 55 rotor (Beckman) at 4 °C. Fractions (70 μl) were collected from the bottom of the tube and analyzed by SDS-polyacrylamide gel electrophoresis. Gel bands were stained with Colloidal Blue (NOVEX) and quantified using NIH image 1.60 software. The sedimentation coefficient was determined relative to those of standard proteins sedimented in parallel gradients. Deoxycytidyl transferase assays were performed as described previously (12Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar, 17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar, 18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) with the oligonucleotides 5′-CTCGTCAGCATCTTCAXCATACAGTCAGTG-3′ (X = G; 30G, A; 30A, T; 30T, C; 30C, U; 30U) and 5′-CTCGTCAGCATCTTCXCCATACAGTCAGTG-3′ (X = G; 30CG, A; 30C, T; 30CT, C; 30CC) as templates and 5′-CACTGACTGTAT-3′ (P12), 5′-CACTGACTGTATG-3′ (P13), and 5′-CACTGACTGTATGX-3′ (X = G; P13G, A; P13A, T; P13T, C; P13C) as oligonucleotide primers. The primers were labeled using polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (Amersham Biosciences, Inc.) and annealed to the templates. To generate an AP site-containing substrate, a primer-template containing a deoxyuracil residue was treated with 0.1 units of Escherichia coli uracil-DNA glycosylase (New England Biolabs) at 30 °C for 30 min in a reaction mixture just before the addition of enzyme. The reaction mixture (25 μl) contained 25 mm potassium phosphate buffer, pH 7.4, 5 mm MgCl2, 0.1 mg/ml BSA, 10% glycerol, 5 mm dithiothreitol, 0.1 mmdNTP, 100 nm primer-template, and 1 μl of protein sample diluted with buffer A containing 0.1 mg/ml BSA as indicated. After incubation at 30 °C for the indicated time, reactions were terminated with 10 μl of stop solution (30 mm EDTA, 94% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol), and products were resolved on 20% polyacrylamide gels containing 8 murea and autoradiographed at −80 °C. The amount of DNA present in each band was quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.). The cloned mouseRev1 cDNA encodes a putative protein of 1249 amino acid residues with a calculated molecular mass of 137 kDa. The sequence alignment of the human and mouse Rev1 proteins is shown in Fig.1. Comparison of the amino acid sequences of the two proteins revealed an overall amino acid identity of 84% and similarity of 90% with all of the motifs found in the human REV1 protein conserved in the mouse counterpart (Fig. 1). The BRCT domain, motif I, and motif VIII are specific to the Rev1 family. Motifs II–VII are conserved in polymerases of the UmuC superfamily. We (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) previously showed the minimum region required for deoxycytidyl transferase activity of the human REV1 protein (Fig. 1, boxed region). This region was highly conserved with 88% identity and 94% similarity. Expression of the mouse Rev1 gene in various tissues was examined by Northern blot analysis (Fig. 2), and the mouse Rev1 mRNA was detected in all tissues examined. Expression of the Rev1 gene was relatively high in the heart, skeletal muscle, and testis. To purify the mouse Rev1 protein, we expressed a recombinant protein tagged with hexa-histidine at its N terminus inE. coli cells. The tagged Rev1 protein (h6-mRev1) was purified by affinity chromatography on a nickel-chelating column, and the fraction containing h6-mRev1 was applied to a gel filtration column. The h6-mRev1 protein eluted with an apparent molecular mass of 330 kDa with a Stokes' radius of 58 Å (Table I). As shown in Fig. 3, analysis by SDS-PAGE revealed a full-length h6-mRev1 protein of 139 kDa, and smaller forms were also detected specifically when the h6-mRev1 protein was induced in E. coli cells, indicating that these bands are degradation products (data not shown). The properties were found to be identical with those of the human REV1 protein (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this preparation, neither DNA polymerase activity nor deoxyribonuclease activity was detected (Fig. 4Aand data not shown). We used this fraction for further physicochemical and biochemical characterization.Table IPhysicochemical properties of the h6-mRev1 proteinProperty1-aThe Stokes' radius was determined by gel filtration using the size markers ferritin (61.0 Å), aldolase (48.1 Å), ovalbumin (30.5 Å), and ribonuclease A (16.4 Å), and the data were based on A280 values monitored during the chromatography. The sedimentation coefficient was determined with catalase (11.3 S), aldolase (7.3 S), and albumin (4.3 S) size markers, and the data were based on the both of SDS-gel profile and dCMP transferase activity. The dCMP transferase activity paralleled exactly the abundance of the h6-mRev1 protein. The molecular mass was calculated from the Stokes' radius and the sedimentation coefficient assuming a partial specific volume of 0.73 (22).Stokes' radius58 ÅSedimentation coefficient (S20,w)5.7 × 10−13 secMolecular mass138 kDa1-a The Stokes' radius was determined by gel filtration using the size markers ferritin (61.0 Å), aldolase (48.1 Å), ovalbumin (30.5 Å), and ribonuclease A (16.4 Å), and the data were based on A280 values monitored during the chromatography. The sedimentation coefficient was determined with catalase (11.3 S), aldolase (7.3 S), and albumin (4.3 S) size markers, and the data were based on the both of SDS-gel profile and dCMP transferase activity. The dCMP transferase activity paralleled exactly the abundance of the h6-mRev1 protein. The molecular mass was calculated from the Stokes' radius and the sedimentation coefficient assuming a partial specific volume of 0.73 (22Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar). Open table in a new tab Figure 4Transferase activity of the h6-mRev1 protein.A, primer extension assays. A 5′-32P-labeled primer, P13, was annealed with each of the templates, 30G (panel a), 30A (panel b), 30T (panel c), 30C (panel d), and 30U (panel e). The primer-template containing an AP site (panel f) was generated as described under "Experimental Procedures." Thenucleotide sequences adjacent to the primer terminus are shown on the right of each panel. Ten ng of the h6-mRev1 protein and the indicated primer-template were incubated with no dNTP (−), a single dNTP (G, A, T, C), or all four dNTPs (N) at 30 °C for 30 min in a 25-μl reaction solution. The reaction products were resolved in 20% polyacrylamide gels containing 8 m urea and visualized by autoradiography. B, analysis of the products from the dGMP and dTMP transfer reactions. Reaction products from the G template with dGTP (lane 1), dTTP (lane 6), and dCTP (lane 7), shown in panel a ofA, were loaded on a gel with the 5′-32P-labeled oligonucleotide markers P13G (lane 2), P13A (lane 3), P13C (lane 4), and P13T (lane 5).C, quantitation of results of primer extension assays. The band intensities of substrates and products were determined using a Bio-Imaging Analyzer BAS2000, and the amounts (in pmol) of products in a 25-μl reaction solution were calculated.D, transferase activity of the recombinant human REV1 protein (h6-hREV1). A 5′-32P-labeled primer, P13, was annealed with the template, 30G. The nucleotide sequencesadjacent to the primer terminus are shown on the right of the panel. Ten-ng aliquots of the h6-hRev1 protein or a mutant protein, D569A/E570A, were incubated with no dNTP (−) or a single dNTP (G, A, T, C) at 30 °C for 30 min in a 25-μl reaction solution.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the molecular mass of the purified h6-mRev1 protein in solution, it was analyzed by sucrose gradient sedimentation. The determined sedimentation coefficient was 5.7 S. Employing the method described by Siegel and Monty (22Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar), we calculated the molecular mass at 138 kDa (Table I). These results suggested that the h6-mRev1 protein was asymmetrically shaped, existing as a monomer in solution. Using six different primer-templates, we examined the substrate specificity of the transferase activity of the h6-mRev1 protein in the presence of 100 μm dGTP, dATP, dTTP, or dCTP (Fig. 4). In this experiment, the respective primer-templates differed only at the template nucleotide immediately downstream from the annealed primer. When a G template was incubated with the h6-mRev1 protein and each of the dNTPs, we surprisingly detected a one-base-extended product in the presence of not only dCTP but also dGTP and dTTP (Fig. 4A, panel a). We also found an ability to insert dGMP and dTMP residues opposite the template AP site (Fig. 4A, panel f). Although the efficiency was very low, significant activity was confirmed by reactions with higher concentrations of the h6-mRev1 protein (data not shown). We could not detect dGMP insertion opposite template C or dTMP insertion opposite template A (Fig. 4A, panels b andd), and the results clearly indicated that activity from contaminating bacterial DNA polymerases was less than the detectable level. The mobility of the reaction products and defined oligonucleotide markers was compared (Fig. 4B). Those with dGTP, dTTP, and dCTP migrated exactly like the respective markers (Fig.4B). These results indicate that the h6-mRev1 protein has a potential to transfer dGMP and dTMP residues, albeit with only a fifth of the activity with dCMP residues (Fig. 4C). In the presence of all four dNTPs, the dCMP transfer reaction predominated over the dGMP and dTMP transfer reactions (Fig. 4A,panel a, lane 6). Only the ability to transfer dCMP has been reported for the human REV1 protein (17Lin W. Xin H. Zhang Y. Wu X. Yuan F. Wang Z. Nucleic Acids Res. 1999; 27: 4468-4475Crossref PubMed Scopus (164) Google Scholar, 18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, when dGMP and dTMP transferase activities of the human REV1 protein were examined (Fig. 4D), it incorporated not only dCMP but also dGTP and dTTP residues opposite template G. The activity was completely eliminated with an inactive mutant protein, D569A/E570A (18Masuda Y. Takahashi M. Tsunekuni N. Minami T. Sumii M. Miyagawa K. Kamiya K. J. Biol. Chem. 2001; 276: 15051-15058Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) (Fig. 4D). When all of the templates were tested, the h6-mRev1 protein inserted a dCMP residue opposite not only the template AP site but also all bases examined (Fig. 4A,panels b–f, lane 5). The enzyme activity for insertion reactions opposite the template AP site was slightly higher than that opposite templates G, A, and U (1.2–1.5-fold) and six times higher than that opposite templates T and C (Fig. 4C). When G, A, T, and U templates were incubated with h6-mRev1 and dCTP, a faint two base longer band was detected (Fig. 4A,panels a–c and e), and it was concluded that the h6-mRev1 protein might possess the ability to add a dCMP residue to the mismatched 3′ terminus resulting from the first insertion of a dCMP residue. We investigated this preliminary observation further by performing reactions using another set of primer-templates having mismatched primer-template termini (Fig.5, A and B) differing only in the attachment of a cytidine residue at the 3′ terminus from the set of primer-templates described in Fig. 4. As illustrated in Fig. 5, A and B, the transferase extended C·A, C·T, and C·U (primer·template) mismatched termini by only one nucleotide (Fig. 5A,panels b, c, and e) but failed to extend C·C and C·AP (primer·template) termini (Fig.5A, panels d and f). The ability did not arise from a potential of the enzyme to extend the 3′ terminus of the single-stranded DNA (Fig. 5C). In the sequence context, it should be noted that the enzyme activity for extension of the mismatched primer termini was 10 times lower than that of the matched C·G primer (primer·template) terminus (Fig. 5B and TableII).Table IIExtension activity with matched and mismatched 3′ terminiPrimer/template2-a5′-32P-labeled P13G, P13A, P13T, and P13C primers were each annealed with the templates: 30G, 30A, 30T, and 30C. The base pairs in the primer terminus next to the primer/template are indicated in parentheses.Specific activity2-bAn appropriate amount of h6-mRev1 was incubated with the indicated primer-template and dCTP at 30 °C for 30 min in 25 μl of reaction solution. The reaction products were resolved in 20% polyacrylamide gels containing 8 m urea, and the band intensities of substrates and products were determined using a Bio-Imaging Analyzer BAS2000 to give amounts in picomoles.Relative activitypmol/μg P13G/30G (G·G)4.616 30A (G·A)0.93 30T (G·T)0.72 30C (G·C)28.2100 P13A/30G (A·G)7.213 30A (A·A)2.95 30T (A·T)55.2100 30C (A·C)1.32 P13T/30G (T·G)10.936 (90)2-cSpecific activity of the T·G (primer·template) mismatch-extension reaction was compared with that for the C·G primer terminus. 30A (T·A)30.1100 30T (T·T)1.24 30C (T·C)2.38 P13C/30G (C·G)12.1100 (100)2-cSpecific activity of the T·G (primer·template) mismatch-extension reaction was compared with that for the C·G primer terminus. 30A (C·A)1.08 30T (C·T)0.76 30C (C·C)<0.1<12-a 5′-32P-labeled P13G, P13A, P13T, and P13C primers were each annealed with the templates: 30G, 30A, 30T, and 30C. The base pairs in the primer terminus next to the primer/template are indicated in parentheses.2-b An appropriate amount of h6-mRev1 was incubated with the indicated primer-template and dCTP at 30 °C for 30 min in 25 μl of reaction solution. The reaction products were resolved in 20% polyacrylamide gels containing 8 m urea, and the band intensities of substrates and products were determined using a Bio-Imaging Analyzer BAS2000 to give amounts in picomoles.2-c Specific activity of the T·G (primer·template) mismatch-extension reaction was compared with that for the C·G primer terminus. Open table in a new tab The mismatch-extending ability was examined using all 16 possible combinations of primer-template termini (Fig.6). As shown in Fig. 6, extensions of all mismatched termini except for C·C were detected. The quantitative results are summarized in Table II. Interestingly, the 3′ mismatched termini with a G template, G·G, A·G, and T·G (primer·template) tended to be extended