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Translesion DNA Synthesis Catalyzed by Human Pol η and Pol κ across 1,N 6-Ethenodeoxyadenosine

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m102158200

ISSN

1083-351X

Autores

Robert L. Levine, Holly Miller, Arthur P. Grollman, Eiji Ohashi, Haruo Ohmori, Chikahide Masutani, Fumio Hanaoka, Masaaki Moriya,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

1,N 6-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) η and κ, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol η than with pol κ and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol η, may contribute to the translesion DNA synthesis events observed for 1,N 6-ethenodeoxyadenosine in human cells. 1,N 6-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) η and κ, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol η than with pol κ and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol η, may contribute to the translesion DNA synthesis events observed for 1,N 6-ethenodeoxyadenosine in human cells. DNA polymerase translesion DNA synthesis 1,N 6-ethenodeoxyadenosine proliferating cell nuclear antigen dithiothreitol bovine serum albumin In the last few years, several new human DNA polymerases (pols),1 which are likely to be involved in translesion DNA synthesis (TLS), were discovered. This list includes pol η (1Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (389) Google Scholar), pol κ (2Ogi T. Kato Jr., T. Kato T. Ohmori H. Genes Cells. 1999; 4: 607-618Crossref PubMed Scopus (142) Google Scholar, 3Gerlach 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), pol ι (4McDonald J.P. Rapic-Otrin V. Epstein J.A. Broughton B.C. Wang X. Lehmann A.R. Wolgemuth D.J. Woodgate R. Genomics. 1999; 60: 20-30Crossref PubMed Scopus (182) Google Scholar), and pol ζ (5Gibbs P.E. McGregor W.G. Maher V.M. Nisson P. Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6876-6880Crossref PubMed Scopus (296) Google Scholar). Pol η, pol ι, and pol κ are encoded by thehRAD30A, hRAD30B, and DINB1genes, respectively. These new pols form a Rad30/UmuC/DinB/REV1 superfamily (2Ogi T. Kato Jr., T. Kato T. Ohmori H. Genes Cells. 1999; 4: 607-618Crossref PubMed Scopus (142) Google Scholar, 3Gerlach 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, 6Johnson R.E. Washington M.T. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12224-12226Crossref PubMed Scopus (131) Google Scholar). Pol ζ consists of two gene products: hREV3 containing pol activity and hREV7 with an unknown function (5Gibbs P.E. McGregor W.G. Maher V.M. Nisson P. Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6876-6880Crossref PubMed Scopus (296) Google Scholar). In general, these pols catalyze TLS more efficiently than previously known pols. They synthesize DNA in a distributive manner and tend to show lower replication fidelity than other pols such as pol α, pol β, and pol δ when unmodified DNA is used as a template (7Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 8Matsuda T. Bebenek K. Masutani C. Hanaoka F. Kunkel T.A. Nature. 2000; 404: 1011-1013Crossref PubMed Scopus (331) Google Scholar, 9Ohashi E. Bebenek K. Matsuda T. Feaver W.J. Gerlach V.L. Friedberg E.C. Ohmori H. Kunkel T.A. J. Biol. Chem. 2000; 275: 39678-39684Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 10Tissier A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar). There are several pieces of evidence for the involvement of human pol η and pol ζ in TLS in vivo (5Gibbs P.E. McGregor W.G. Maher V.M. Nisson P. Lawrence C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6876-6880Crossref PubMed Scopus (296) Google Scholar, 11Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Crossref PubMed Scopus (531) Google Scholar, 12Wang Y.C. Maher V.M. McCormick J.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7810-7814Crossref PubMed Scopus (64) Google Scholar, 13Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1162) Google Scholar). Although the involvement of human pol κ or pol ι has not yet been establishedin vivo, the Escherichia coli homologue of pol κ, pol IV, has been shown to play a role in TLS in vivo(14Napolitano R. Janel-Bintz R. Wagner J. Fuchs R.P. EMBO J. 2000; 19: 6259-6265Crossref PubMed Scopus (330) Google Scholar). Pol η, which is missing in xeroderma pigmentosum variant cells (13Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1162) Google Scholar, 15Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (676) Google Scholar), is shown to catalyze efficient TLS across thecis-syn cyclobutane thymine-thymine dimer by inserting two dAMPs opposite the lesion (16Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; 19: 3100-3109Crossref PubMed Google Scholar). Therefore, the cancer proneness of xeroderma pigmentosum variant patients is thought to be caused by the lack of accurate TLS across this and/or other UV photo products. Pol η also catalyzes TLS across other DNA lesions such as cisplatin G-G intrastrand cross-link (16Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; 19: 3100-3109Crossref PubMed Google Scholar), acetylaminofluorene-dG (16Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; 19: 3100-3109Crossref PubMed Google Scholar), and 8-oxodeoxyguanosine (17Haracska L., Yu, S.L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (309) Google Scholar) with relatively high fidelity. On the other hand, TLS across (+)-trans-anti-benzo[a]pyrene-N 2-dG is reported to be error-prone (18Zhang Y. Yuan F. Wu X. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4717-4724Crossref PubMed Google Scholar). Pol κ is also shown to conduct TLS across an abasic site (19Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar, 20Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Crossref PubMed Google Scholar), acetylaminofluorene-dG (19Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar,20Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Crossref PubMed Google Scholar), (−)-trans-anti-benzo[a]pyrene-N 2-dG (20Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Crossref PubMed Google Scholar), and 8-oxodeoxyguanosine (20Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Crossref PubMed Google Scholar). Based on these findings, we are motivated to study the efficiency and fidelity of TLS catalyzed by these novel pols across 1,N 6-ethenodeoxyadenosine (εdA) to probe thein vivo TLS mechanism. We have shown that εdA is miscoding in simian and human cells by inducing εdA→T, εdA→G, and εdA→C (21Pandya G.A. Moriya M. Biochemistry. 1996; 35: 11487-11492Crossref PubMed Scopus (184) Google Scholar, 22Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). This adduct is produced in animals exposed to vinyl compounds such as the human carcinogen vinyl chloride. Surprisingly, this adduct is also found in unexposed animals and humans with lipid peroxidation products being the suspected source of this adduct (23Singer B. Bartsch H. Exocyclic DNA Adducts in Mutagenesis and Carcinogenesis. International Agency for Research on Cancer, Lyon, France1999: 150Google Scholar). Our in vitro primer extension studies indicate that pol η catalyzes TLS more efficiently than pol κ and that both pols catalyze error-free and error-prone TLS. [γ-32P]ATP was purchased from Amersham Pharmacia Biotech. Human pol η and pol κ were purified as described (13Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1162) Google Scholar, 19Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar). Pol δ was purified to apparent homogeneity from calf thymus (24Ng L. Tan C.K. Downey K.M. Fisher P.A. J. Biol. Chem. 1991; 266: 11699-11704Abstract Full Text PDF PubMed Google Scholar). Human proliferating cell nuclear antigen (PCNA) was a generous gift from Paul Fisher (State University of New York, Stony Brook, NY). T4 polynucleotide kinase and EcoRI were purchased from New England Biolabs. Ultrapure deoxyribonucleic acid triphosphates were purchased from Roche Molecular Biochemicals. Oligonucleotides were purchased from Oligos Etc. (Wilsonville, OR) or synthesized in the laboratory of Francis Johnson (State University of New York, Stony Brook, NY). Oligomers were purified by electrophoresis on a 20% polyacrylamide gel containing 7 m urea, detected by UV shadowing, excised from the gel, eluted from gel slices, and desalted using a SEP-PAK C18 cartridge (Waters). Purified oligonucleotide primers were labeled at the 5′ end with [γ-32P]ATP and T4 polynucleotide kinase. Primers were annealed to templates by mixing at a 1:1.2 molar ratio in 10 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 100 mm NaCl by heating to 80 °C followed by slow cooling. For primer extension and standing start kinetic studies (25Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (227) Google Scholar) of nucleotide insertion and extension, the 32P-labeled primers (5′-GTTCTAGCGTGTAGGT, 5′-GTTCTAGCGTGTAGGTAT, and 5′-GTTCTAGCGTGTAGGTATN (where N = A, C, G, or T)) were annealed to a 28-mer template (5′-CTGCTCCTCXATACCTACACGCTAGAAC (where X = dA or εdA)), generating substrates 1, 2, and 3, respectively. To quantify various TLS products, the 38-mer template (5′-CATGCTGATGAATTCCTTCXCTACTTTCCTCTCCATTT (where X = dA or εdA; EcoRI site shown in bold)) was annealed to the32P-labeled primer (5′-AGAGGAAAGTAG), yielding substrate 4 (Fig. 2). Each reaction mixture (10 μl) contained 40 nm substrate 1 and 100 μm dNTPs. Reactions with pol η (1Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (389) Google Scholar) or pol κ (19Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar) contained 40 mmTris-HCl (pH 8.0), 60 mm KCl, 5 mmMgCl2, 10 mm dithiothreitol (DTT), 2.5% glycerol, and 250 μg/ml bovine serum albumin (BSA). Reactions with pol δ contained 7 ng/μl PCNA, 40 mm Bis-Tris (pH 6.8), 6 mm MgCl2, 2 mm DTT, 4% glycerol, and 40 μg/ml BSA. Pol η was diluted in 20 mmpotassium phosphate (pH 7.5), 0.3 m KCl, 0.1 mmEDTA, 0.1 mg/ml BSA, 1 mm DTT, and 50% glycerol. Pol κ was diluted in 10 mm Tris-HCl (pH 7.4), 0.3 mKCl, 1 mm EDTA, 0.2 mg/ml BSA, 1 mm DTT, and 50% glycerol. Pol δ was diluted in 40 mm Bis-Tris (pH 6.8), 1 mm DTT, 0.2 mg/ml BSA, and 10% glycerol. Reactions were initiated by adding enzyme and were incubated at 37 ± 1 °C for 10 min (pol η or pol κ) or 30 ± 1 °C for 30 min (pol δ). Reactions were stopped by adding 10 μl of 95% formamide dye mixture (95% formamide, 10 mm EDTA, 0.001% xylene cyanol, and 0.001% bromphenol blue), and then the mixture was heated to 95 °C for 5 min. Aliquots (1 μl) were subjected to electrophoresis in a denaturing 20% polyacrylamide gel. Standing-start reactions (25Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (227) Google Scholar) (10 μl) contained 40 nm substrate 2 or 3 (substrate 2 for insertion analysis and substrate 3 for extension analysis), 0–2 mm dNTP(s), and a reaction buffer (see above). Initiation and termination of reactions were conducted as described above. Aliquots (1 μl) were subjected to electrophoresis in a denaturing 20% polyacrylamide gel. Integrated gel band intensities were measured using a PhosphorImager and ImageQuant software (Molecular Dynamics). Nucleotide incorporation parameters were determined (25Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (227) Google Scholar). Less than 20% of the primers were extended in these steady-state kinetic analyses, ensuring single-hit kinetics (26Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (407) Google Scholar). Values for the Michaelis-Menten constant (K m) andV max for incorporation opposite dA and εdA were obtained by least squares nonlinear regression to a rectangular hyperbola. k cat was calculated by dividingV max by the enzyme concentration. The frequency of insertion (F ins) and extension (F ext) were calculated using the equationF ins or ext = (k cat/K m)adduct/(k cat/K m)control(25Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (227) Google Scholar). Standard errors derived from the curve-fitting are included. DNA synthesis reaction mixtures (10 μl) contained 50 nm substrate 4, 100 μmdNTPs, the appropriate buffer (see above), and enzyme (1.5 units of pol δ, 36 fmol of pol η, or 56 fmol pol κ) were incubated at 23 ± 1 °C for 15 min and then 37 ± 1 °C for 45 min (27Shibutani S. Suzuki N. Matsumoto Y. Grollman A.P. Biochemistry. 1996; 35: 14992-14998Crossref PubMed Scopus (59) Google Scholar). Reactions were stopped by adding 10 μl of a formamide dye mixture and heating to 95 °C for 5 min. Samples were subjected to electrophoresis in a denaturing 20% polyacrylamide gel (35 × 42 × 0.04 cm). Full-length products were extracted from the gel and annealed to a complementary 38-mer. The annealed products were digested with EcoRI (100 units) for 1 h at 30 °C and then 1 h at 15 °C. This digestion generates32P-labeled 18-mers from the fully extended products. The products were separated in a two-phase polyacrylamide gel (15 × 72 × 0.04 cm) (27Shibutani S. Suzuki N. Matsumoto Y. Grollman A.P. Biochemistry. 1996; 35: 14992-14998Crossref PubMed Scopus (59) Google Scholar). This method allows the separation of four base substitution products and frameshift products. The DNA template of substrate 4 is different from the template of substrates 1, 2, and 3 in the DNA sequence surrounding εdA. The sequence context used in the template of substrates 1, 2, and 3 is identical to that used in miscoding studies in human cells (22Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). It was not possible to separate various TLS products by the method described above when this sequence context was employed. Therefore, we used the sequence (substrate 4) that has been shown to permit separation of various TLS products by gel electrophoresis (27Shibutani S. Suzuki N. Matsumoto Y. Grollman A.P. Biochemistry. 1996; 35: 14992-14998Crossref PubMed Scopus (59) Google Scholar). Pol η, pol κ, and pol δ/PCNA were assayed for polymerase activity on both unmodified and εdA-modified templates. The primer (substrate 1; Fig.1 A) allowed the addition of two nucleotides before encountering the adduct. Although all three pols were capable of synthesizing across εdA (Fig. 1, B andC), this lesion posed a much stronger block to pol δ than to pol η and pol κ when compared with the control templates. A very small amount of the full-length product was generated by pol δ only when PCNA was added to the reaction mixture (compare lanes 10 and 11 with lane 12 in Fig.1 B), revealing the enhancing role for PCNA in TLS. Pol η seems to catalyze TLS more efficiently than pol κ. To determine the efficiency and fidelity of TLS catalyzed by pol η and pol κ, we first determined steady-state kinetic parameters (K m and k cat) for nucleotide incorporation opposite dA and εdA using substrate 2. The internal 13 nucleotides (5′-CTCCTCXATACCT) of this template are identical to those used in the miscoding studies in human cells (22Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar). The kinetic data (F ins) indicate that pol η incorporates a nucleotide opposite εdA more efficiently than pol κ. Pol η inserts the correct dTMP opposite εdA twice as efficiently as dAMP and dGMP and 13 times more efficiently than dCMP. This dTMP insertion is ∼68 times less efficient than that opposite dA. Similarly, pol κ also inserts dTMP most efficiently opposite εdA, followed by dGMP and then dAMP, but its efficiency is ∼1000 times less than the incorporation opposite dA. These results indicate that dTMP, the correct nucleotide, is preferentially inserted opposite εdA by both pols. We then determined steady-state kinetic parameters for nucleotide extension from four different 3′ termini located opposite dA or εdA using substrate 3. The kinetic data (F ext) indicate that pol η extends from all the termini more efficiently than pol κ. Pol η extends the primer with the correct dTMP terminus more efficiently than the other three termini when the modified template was used. This extension from the dTMP terminus is ∼55 times less efficient than that from the dTMP terminus located opposite dA. In experiments using pol κ, the efficiency of extension from the 3′ terminus followed the order of dAMP > dGMP > dTMP > dCMP, indicating that unlike pol η, the incorrect pairings are extended better than the correct εdA:T pairing. Based on these insertion and extension kinetic parameters, the relative efficiency of TLS was determined by multiplyingF ins and F ext. The results indicate that pol η catalyzes TLS across εdA more efficiently than pol κ. With pol η, TLS with εdA:T is dominant, and its efficiency is 3.0, 4.4, and 45 times greater than TLS with εdA:A, εdA:G, and εdA:C, respectively. The same analysis for pol κ shows that the efficiency of TLS with εdA:T is 2.1 and 2.7 times greater than TLS with εdA:A and εdA:G, respectively. These results indicate that accurate TLS is dominant but not exclusive with both pols. Because steady-state kinetic analysis includes only one of four dNTPs in the reaction mixture, and frameshift mutations are not detected, we determined the miscoding specificity of εdA in the presence of four dNTPs. We analyzed polymerization products using substrate 4 (Fig.2). Although fully extended products were observed with the unmodified template for all three pols, only pol η and pol κ produced full-length products with the modified template (data not shown). One possible explanation is that the 10-mer primer is not long enough to accommodate both pol δ and PCNA (28Mozzherin D.J. Tan C.K. Downey K.M. Fisher P.A. J. Biol. Chem. 1999; 274: 19862-19867Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and pol δ alone cannot catalyze TLS as shown in Fig. 1 B. Fully extended products were digested with EcoRI and electrophoresed in a two-phase polyacrylamide gel. When the unmodified template was used, pol δ-catalyzed products showed only one band (Fig. 2, lane 2) that co-migrated with the dT marker, indicating accurate DNA synthesis. Pol κ also catalyzed faithful synthesis (Fig. 2, lane 5). Pol η mainly catalyzed error-free DNA synthesis, but two additional bands were also observed, co-migrating with the dG and two-base deletion standards (Fig. 2,lane 3), indicating that pol η produced errors on the unmodified template. When the modified template was used, at least five and four products were observed for pol η and pol κ, respectively (Fig. 2, lanes 4 and 6), co-migrating with the dG, dA, dT, dC, or one-base deletion markers (Fig. 2, lanes 1 and 7). These products were quantified based on the amount of radioactivity in the bands (Table II). Consistent with the results of the steady-state kinetic analysis, pol η dominantly catalyzed accurate TLS with dT on the modified template. However, substantial amounts of products containing dA, dG, dC, or one-base deletion were also observed. On the other hand, pol κ dominantly catalyzed TLS with one-base deletion, followed by dT, dA, and dC incorporation. The results shown in Table II indicate that error-prone TLS is dominant for both pols when frameshift mutations are included and that pol η catalyzes accurate TLS more frequently than pol κ, but still more than 50% of the TLS is error-prone.Table IIMiscoding properties of ɛdA in reactions catalyzed by pol δ, pol η, or pol κEnzymeLesiondAdCdGdTΔ1Δ2Pol δdA00010000Pol ηdA2.90.911.476.81.06.9Pol ηɛdA19.84.79.142.719.91.9Pol κdA01.4096.600.5Pol κɛdA18.84.52.930.245.60.9Numbers represent amounts of products expressed as a percentage of a total amount of fully extended products. Δ1, one-base deletion; Δ2, two-base deletion. Open table in a new tab Numbers represent amounts of products expressed as a percentage of a total amount of fully extended products. Δ1, one-base deletion; Δ2, two-base deletion. Pol η has been reported to catalyze TLS across several DNA lesions in a relatively error-free manner (1Masutani C. Araki M. Yamada A. Kusumoto R. Nogimori T. Maekawa T. Iwai S. Hanaoka F. EMBO J. 1999; 18: 3491-3501Crossref PubMed Scopus (389) Google Scholar, 16Masutani C. Kusumoto R. Iwai S. Hanaoka F. EMBO J. 2000; 19: 3100-3109Crossref PubMed Google Scholar, 17Haracska L., Yu, S.L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (309) Google Scholar). Our steady-state kinetic analyses and the analysis of TLS products have revealed that TLS catalyzed by pol η across εdA, like the benzo[a]pyrene dG adduct (18Zhang Y. Yuan F. Wu X. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4717-4724Crossref PubMed Google Scholar), is significantly erroneous. Although accurate TLS with dTMP insertion opposite εdA is predominant, pol η also frequently catalyzed erroneous TLS, causing base substitutions and frameshift mutations (Tables I and II and Fig. 2). Pol κ catalyzes TLS across several DNA lesions in relatively error-free and error-prone manners (19Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594PubMed Google Scholar, 20Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Crossref PubMed Google Scholar). Our steady-state kinetic analysis shows that pol κ preferentially incorporates dTMP, followed by dAMP, opposite εdA (Table I). The product analysis experiment (Table II and Fig. 2), however, has revealed that one-base deletion events were dominant followed by dTMP insertion products, indicating that pol κ-catalyzed TLS is also erroneous. Neither pol η nor pol κ can be characterized as simply error-free or error-prone polymerases.Table IKinetic parameters for nucleotide insertion and chain extension catalyzed by pol η and pol κPol η N:XInsertionExtensionF ins× F extK mk catk cat/K mF insK mk catk cat/K mF extμmmin−1μmmin−1T:dA21.4 ± 5.26.2 ± 0.60.291.00.67 ± 0.137.5 ± 0.411.21.01.0A:ɛdA225 ± 360.49 ± 0.032.2 × 10−37.4 × 10−37.0 ± 1.20.94 ± 0.030.1312.1 × 10−39.0 × 10−5C:ɛdA493 ± 550.16 ± 0.013.0 × 10−41.1 × 10−32.2 ± 0.50.14 ± 0.010.0655.8 × 10−30.6 × 10−5G:ɛdA42.7 ± 9.60.082 ± 0.0051.9 × 10−36.6 × 10−313.7 ± 5.01.4 ± 0.10.109.3 × 10−36.1 × 10−5T:ɛdA65.7 ± 23.60.28 ± 0.044.3 × 10−314.7 × 10−33.5 ± 0.50.72 ± 0.020.2018.3 × 10−326.9 × 10−5Pol κ N:XInsertionExtensionF ins× F extK mk catk cat/K mF insK mk catk cat/K mF extT:dA9.4 ± 0.911.19 ± 0.361.21.01.8 ± 0.341.8 ± 2.423.21.01.0A:ɛdA455 ± 960.081 ± 0.0062.0 × 10−41.5 × 10−4197 ± 444.4 ± 1.00.0239.7 × 10−41.5 × 10−7C:ɛdANDNDNDND224 ± 1060.56 ± 0.290.00251.1 × 10−4<0.1 × 10−7G:ɛdA126 ± 290.034 ± 0.0023.0 × 10−42.3 × 10−4287 ± 413.5 ± 0.40.0125.3 × 10−41.2 × 10−7T:ɛdA291 ± 690.34 ± 0.031.2 × 10−39.9 × 10−4110 ± 300.83 ± 0.200.00763.3 × 10−43.2 × 10−7ND, not determined. Open table in a new tab ND, not determined. The overall efficiency of TLS, determined byF inc × F ext, for A:εdA and G:εdA is similar for both pols: 9.0 × 10−5 versus 6.1 × 10−5 for pol η and 1.5 × 10−5 versus 1.2 × 10−5 for pol κ (Table I). However, analysis of TLS products shows that dA incorporation is preferred to dG by both pols: 19.8% dA versus 9.1% dG for pol η and 18.8% dAversus 2.9% dG for pol κ (Table II). It is likely that some dGMP incorporated opposite εdA misaligned (Fig.3, step 3) to generate a one-base deletion, whereas dAMP incorporation does not cause this misalignment. Accordingly, dAMP incorporation opposite εdA leads to a base substitution, whereas dGMP incorporation results in both a base substitution and a one-base deletion. Another mechanism envisioned is "dNTP-stabilized misalignment," which was observed for pol β in TLS across abasic sites (29Efrati E. Tocco G. Eritja R. Wilson S.H. Goodman M.F. J. Biol. Chem. 1997; 272: 2559-2569Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). According to this mechanism, the slippage event occurs first, causing εdA to be extrahelical (step 2), and the incoming dGTP stabilizes this misalignment (step 4). Continuous extension from this terminus results in a one-base deletion (step 5), whereas realignment (step 7) and extension (step 8) result in a base substitution. The hallmark of the dNTP-stabilized misalignment mechanism is the relatively low K m for dNMP insertion at the terminus opposite a DNA lesion, which suggests that the incorporation is actually opposite the base 5′ to the lesion (29Efrati E. Tocco G. Eritja R. Wilson S.H. Goodman M.F. J. Biol. Chem. 1997; 272: 2559-2569Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In the insertion kinetic studies with pol η, theK m value for dGMP insertion opposite εdA (42.7 μm) is not much different from that for dTMP insertion opposite dA (21.4 μm) (Table I), which suggests that dGMP is inserted opposite dC, 5′ to εdA, and extension from this terminus results in a one-base deletion (steps 2 to 4 to5). On the other hand, with pol κ theK m value for dGMP insertion opposite εdA (126 μm) is very different from that for dTMP insertion opposite dA (9.4 μm) (Table I). This suggests that dGMP is incorporated opposite εdA, followed by misalignment and subsequent extension of the primer. This is the likely mechanism for the induction of one-base deletions (the dominant event) by pol κ. Our mutagenesis experiments (22Levine R.L. Yang I.Y. Hossain M. Pandya G.A. Grollman A.P. Moriya M. Cancer Res. 2000; 60: 4098-4104PubMed Google Scholar) have shown that miscoding events account for 10–20% of TLS in human cells. Although it is not possible to speculate as to what extent these pols contribute to TLS in vivo, our results suggest that if these pols are involved in TLSin vivo, then it is likely to be error-prone. In vivo experiments using human cells lacking these pols are necessary to clarify this point. We thank F. Johnson, C. Torres, and S. Shibutani for oligonucleotides used in this research.

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