Inefficient Bypass of an Abasic Site by DNA Polymerase η

DNA polymerase η (Polη) bypasses acis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Polη in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Polη bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O6-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Polη, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Polη are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Polη bypasses this lesion extremely poorly. These results suggest that Polη requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation. DNA polymerase η (Polη) bypasses acis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Polη in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Polη bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O6-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Polη, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Polη are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Polη bypasses this lesion extremely poorly. These results suggest that Polη requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation. apurinic/apyrimidinic thymine-thymine 8-oxoguanine nucleotide(s) polymerase η human Polη yeast Polη Abasic (apurinic/apyrimidinic; AP)1 sites represent one of the most frequently formed DNA lesions in eukaryotic cells. Base loss can occur by spontaneous hydrolysis of the N-glycosylic bond or by the action of DNA glycosylases on damaged bases. It has been estimated that a mammalian cell loses up to 10,000 purines/day from its genome (1Lindahl T. Nyberg B. Biochemistry. 1972; 11: 3610-3617Crossref PubMed Scopus (1180) Google Scholar). In eukaryotes, AP sites are efficiently repaired by excision repair processes (2Ramotar D. Popoff S.C. Gralla E.B. Demple B. Mol. Cell. Biol. 1991; 11: 4537-4544Crossref PubMed Scopus (192) Google Scholar, 3Johnson R.E. Torres-Ramos C.A. Izumi T. Mitra S. Prakash S. Prakash L. Genes Dev. 1998; 12: 3137-3143Crossref PubMed Scopus (183) Google Scholar, 4Torres-Ramos C.A. Johnson R.E. Prakash L. Prakash S. Mol. Cell. Biol. 2000; 20: 3522-3528Crossref PubMed Scopus (71) Google Scholar). However, if not removed, they present a block to the replication machinery. Thus, to maintain the continuity of DNA during replication, AP sites encountered by the replication machinery have to be bypassed. In the yeast Saccharomyces cerevisiae, genes in the RAD6 epistasis group promote replication through DNA lesions (5Prakash L. Mol. Gen. Genet. 1981; 184: 471-478Crossref PubMed Scopus (234) Google Scholar, 6Johnson R.E. Henderson S.T. Petes T.D. Prakash S. Bankmann M. Prakash L. Mol. Cell. Biol. 1992; 12: 3807-3818Crossref PubMed Scopus (196) Google Scholar, 7McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar). The REV1, REV3, andREV7 genes of this epistasis group are essential for damage-induced mutagenesis (8Lawrence C.W. Hinkle D.C. Cancer Surv. 1996; 28: 21-31PubMed Google Scholar), including mutagenesis induced by AP sites (3Johnson R.E. Torres-Ramos C.A. Izumi T. Mitra S. Prakash S. Prakash L. Genes Dev. 1998; 12: 3137-3143Crossref PubMed Scopus (183) Google Scholar). The Rev1 protein has a deoxycytidyltransferase activity that can incorporate a dCMP residue opposite an abasic site (9Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (507) Google Scholar), and the Rev3 and Rev7 proteins associate to form DNA polymerase ζ (10Nelson J.R. Lawrence C.W. Hinkle D.C. Science. 1996; 272: 1646-1649Crossref PubMed Scopus (599) Google Scholar).In vitro, the combination of Rev1 and Polζ promotes AP bypass (9Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (507) Google Scholar). The yeast RAD30 gene, which belongs to the RAD6epistasis group, encodes a DNA polymerase, Polη, that has the unique ability to efficiently replicate through a cis-synthymine-thymine (T-T) dimer; it does so correctly by inserting two A residues across from the T-T dimer (11Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (696) Google Scholar, 12Washington M.T. Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3094-3099PubMed Google Scholar). Human Polη resembles yeast Polη in replicating through the T-T dimer with the same efficiency and accuracy as through undamaged Ts (13Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Consistent with the error-free bypass of the T-T dimer, inactivation of yeast and human Polη causes UV hypermutability (7McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 14Wang Y.-C. Maher V.M. Mitchell D.L. McCormick J.J. Mol. Cell. Biol. 1993; 13: 4276-4283Crossref PubMed Scopus (142) Google Scholar, 15Waters H.L. Seetharam S. Seidman M.M. Kraemer K.H. J. Invest. Dermatol. 1993; 101: 744-748Abstract Full Text PDF PubMed Google Scholar). Patients with the variant form of xeroderma pigmentosum are defective in Polη (16Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (673) Google Scholar, 17Masutani 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 (1153) Google Scholar), and as a consequence, they suffer from a high incidence of UV-induced skin cancers. In addition to the T-T dimer, yeast and human Polη are able to bypass the 8-oxoguanine (8-oxoG) lesion efficiently and accurately (18Haracska L., Yu, S.-L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (306) Google Scholar). In contrast to eukaryotic polymerases α, δ, and ε, which preferentially incorporate an A opposite the 8-oxoG lesion, Polη predominantly inserts a C opposite the 8-oxoG lesion (18Haracska L., Yu, S.-L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (306) Google Scholar). Also, yeast and human Polη are able to bypass the O6-methylguanine (m6G) lesion, and they incorporate a C or a T residue opposite this lesion (19Haracska L. Prakash S. Prakash L. Mol. Cell. Biol. 2000; 20: 8001-8007Crossref PubMed Scopus (118) Google Scholar). For DNA polymerases lacking the proofreading 3′ → 5′ exonuclease activity, the fidelity for nucleotide insertion depends upon the requirement of the polymerase active site for correct Watson-Crick base pairing geometry and upon the ability of bases to form proper hydrogen (H) bonding. Most DNA polymerases are highly sensitive to geometric distortions in DNA (20Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (620) Google Scholar), and their fidelity is affected more severely by the disruption of optimal geometry than by H bonding between base pairs (21Moran S. Ren R.X.-F. Kool E.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10506-10511Crossref PubMed Scopus (301) Google Scholar, 22Goodman M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10493-10495Crossref PubMed Scopus (216) Google Scholar). As a consequence, they are unable to incorporate nucleotides opposite lesions that distort the DNA helix. Previously, we suggested that the ability of Polη to bypass lesions, such as the T-T dimer, 8-oxoG, and m6G, results from an unusual tolerance of its active site for the distorted template geometries of these lesions. To further assess the range of template lesions tolerated by Polη, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. We find that Polη inserts nucleotides opposite the AP site very poorly, and it also extends from the inserted nucleotide very inefficiently. These results suggest that Polη requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide insertion. Standard DNA polymerase reactions (10 μl) contained 40 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin, 10% glycerol, 20 nm 5′32P-labeled oligonucleotide primer annealed to an oligonucleotide template, and dNTP in the concentrations indicated in the figure legends. Reactions were initiated by adding yeast or human Polη at the concentrations indicated in the figure legends. After incubation for 5 min at 30 °C, reactions were terminated by the addition of 40 μl of loading buffer containing 20 mmEDTA, 95% formamide, 0.3% bromphenol blue, and 0.3% cyanol blue. The reaction products were resolved on 10 or 20% polyacrylamide gels containing 8 m urea and were dried before autoradiography at −70 °C with intensifying screens. A Molecular Dynamics STORM phosphorImager and ImageQuant software were used for quantitation. DNA substrates S-1 and S-2 were generated by annealing the 75-nt oligomer template (N75AP, 5′-AGCTACCATGCCTGCCTCAAGAGTTCGTAA0ATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-3′), which contained an AP site (a tetrahydrofuran moiety; Midland Co.) at the underlined 0at position 31 or a nondamaged G residue at this position, respectively, to the 32-nt 5′ 32P-labeled oligomer primer (N4456, 5′-GTTTTCCCAGTCACGACGATGCTCCGGTACTC-3′). For steady-state kinetic analysis, DNA substrates S-3, S-4(G), S-4(A), S-4(T), and S-4(C) were generated by annealing a 52- nt oligomer template (5′-TTCGTATAATGCCTACACT0GAGTACCGGA GCATCGTCGTGACTGGGAAAAC-3′), which contained an AP residue at the underlined position 20 to the 32-nt and four different 33-nt 5′ 32P-labeled oligomer primers (N4456 or oligonucleotides that contain N4456 with one additional G, A, T or C residue at its 3′-end, respectively). DNA substrates S-5(G), S-5(A), S-5(T), and S-5(C) were generated by annealing the N75AP oligomer template to four different 45-nt 5′32P-labeled oligomer primers that contain oligomer N4309 (5′-GTTTTCCCAGTCACGACGATGCTCCGGTACTCCAGTGTAGGCAT-3′) with one additional G, A, T, or C residue at its 3′-end. In nondamaged control DNA substrates the complementary bases were used instead of the AP site. The sequence of the DNA substrate containing the 18-nt template oligomer annealed to the 12-nt primer is shown in the figures. Steady-state kinetic analysis for each deoxynucleotide incorporation opposite the AP site was done as described previously (23Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar, 24Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (404) Google Scholar, 25Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Abstract Full Text PDF PubMed Google Scholar). Analyses of primer extension from this lesion were carried out in a similar manner, except that only the correct incoming deoxynucleotide was added to the reaction and the primer varied at the 3′ primer end. Briefly, Polη was incubated with increasing concentrations of a single deoxynucleotide (0–1000 μm) for 1 min under standard reaction conditions. Gel band intensities of the substrates and products were quantitated by PhosphorImager. The percentage of primer extended was plotted as a function of dNTP concentration, and the data were fit by nonlinear regression using SigmaPlot 5.0 to the Michaelis-Menten equation describing a hyperbola, v = (Vmax × dNTP/(Km + dNTP). Apparent Km andVmax steady-state parameters were obtained from the best fit. To determine whether yeast Polη replicates past an abasic site in template DNA, we used a running start DNA substrate containing a single AP site in a 75-nt template DNA in which the DNA polymerase must synthesize 12 nt before encountering the lesion. DNA synthesis reactions were carried out in the presence of a 4-fold excess of DNA substrate over Polη and from low to higher dNTP concentrations (0.5–50 μm). yPolη replicated through the AP site very poorly, and even at 50 μm dNTP, only ∼5% translesion synthesis occurred (Fig. 1 A, lanes 5–8) compared with synthesis on a template containing a normal G residue (Fig. 1 A, lanes 1–4). Furthermore, yPolη exhibits two strong stall sites, one right before the lesion and the other opposite the lesion, indicating an inhibition of insertion across from the AP site as well as an inhibition of extension from the nucleotide inserted opposite the lesion. A stall site at the position just after the AP site indicates that elongation opposite the 5′ residue next to the AP site is also inhibited. To identify the deoxynucleotide inserted opposite the AP site, we assayed yPolη on an 18-nt template having either a G or an AP site at position 13 from the 3′ end in the template, primed with a 12-nt primer (Fig. 1 B) in the presence of a single or all four nucleotides. As markers, we used the 13- and 18-nt oligomers representing a primer extended by one nucleotide and full-length products, respectively, and containing a C, A, T, or G residue at position 13, which can be distinguished by their on 20% polyacrylamide gels lanes 1–4). To we used high yPolη as well as high dNTP which on the undamaged G template in synthesis to the end of the template DNA (Fig. 1 under these yPolη carried out AP and only nucleotide incorporation opposite the AP site further extension was (Fig. 1 In the presence of all four yPolη inserted a G residue across from the AP site (Fig. 1 only a single nucleotide present a G yPolη also an A, and T was inserted very opposite the AP site (Fig. 1 lanes we the of nucleotide insertion and extension during DNA synthesis past the AP site. To determine the of nucleotide incorporation by we the Km andVmax steady-state kinetic parameters (23Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar, 24Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (404) Google Scholar, 25Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Abstract Full Text PDF PubMed Google for all four incoming opposite a template AP site. For of the kinetic parameters opposite nondamaged template residues were as yPolη was incubated with the DNA substrate and with increasing concentrations of one of the four The of deoxynucleotide incorporation by yPolη opposite an AP site is shown in A. The parameters were and used to the percentage of each nucleotide opposite the AP site yPolη G, A, T, and C opposite the AP site. analysis indicates that yPolη a G opposite the AP site with a higher efficiency than A. yPolη inserts a G opposite the AP site efficiently than the insertion of G opposite C The other nucleotides were inserted even efficiently The efficiency of nucleotide incorporation opposite an AP site to a nondamaged template residue results from a in the Km for dNTP parameters of insertion reactions by yeast by the efficiency of insertion for each dNTP by the of the insertion of all four efficiency the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template opposite abasic an AP opposite nondamaged by the efficiency of insertion for each dNTP by the of the insertion of all four the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template an AP site. in a For lesion bypass to it is after a nucleotide opposite the lesion, a polymerase the primer the lesion. To examine the efficiency of extension past the AP site, the steady-state kinetic parameters of the addition of the next correct deoxynucleotide by yPolη on substrates in which the 3′ terminus of the primer is with an AP site were the of extension from G, A, T, or C with an AP site. the of extension from these different 3′ the Km and were obtained and used to the efficiency of The of extension opposite from the AP site = which indicates that yPolη extends from a G or an A opposite the AP site extends from G or A as the efficiency of extension in both was by that from the opposite nondamaged complementary bases The efficiency of extension from bases opposite an AP site to the extension from bases opposite a nondamaged residue was also of a in the Km for dNTP Hence, yPolη is very inefficient in inserting nucleotides across from an AP site as well as in extending from the nucleotide parameters of extension reactions by yeast by the efficiency of each extension by the of the of all four efficiency the efficiency of extension from a nucleotide opposite the template AP site to the efficiency of extension from the nucleotide opposite from the complementary template from G, A, T, or C opposite abasic an AP from G, A, T, or C opposite nondamaged by the efficiency of each extension by the of the of all four the efficiency of extension from a nucleotide opposite the template AP site to the efficiency of extension from the nucleotide opposite from the complementary template an AP site. in a We also the ability of the human DNA polymerase η to bypass the AP site. bypasses the AP site very inefficiently. also exhibits two stall sites, one right before the AP site and the other opposite the lesion, indicating that is inhibition of deoxynucleotide insertion opposite the AP site as well as inhibition of extension from this lesion not The extremely ability of to bypass an AP site is further in its steady-state Km andVmax kinetic The of insertion of a single deoxynucleotide opposite an AP site and the of addition of the next correct nucleotide to across from the AP site were as a function of deoxynucleotide also inserts a G than an A opposite the AP site. However, inserts these nucleotides opposite the AP site efficiently than opposite the nondamaged complementary of a in the Km for dNTP The and the of deoxynucleotide insertion opposite the AP site by were also extends from the nucleotide inserted opposite the AP site, and the and the of extension from different with the AP site were Thus, human Polη inserts G than A opposite the AP site, it is more efficient at extending from an A opposite the AP site than from a G opposite this lesion. The extension efficiency of is of a in the Km for dNTP parameters of insertion reactions by human by the efficiency of insertion for each dNTP by the of the insertion of all four the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template opposite abasic an AP opposite nondamaged by the efficiency of insertion for each dNTP by the of the insertion of all four the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template an AP site. in a parameters of extension reactions by human by the efficiency of each extension by the of the of all four efficiency the efficiency of extension from a nucleotide opposite the template AP site to the efficiency of extension from the nucleotide opposite the complementary template from G, A, T, or C opposite abasic an AP from G, A, T, or C opposite nondamaged by the efficiency of each extension by the of the of all four the efficiency of extension from a nucleotide opposite the template AP site to the efficiency of extension from the nucleotide opposite the complementary template an AP site. in a Polη is unique eukaryotic DNA polymerases in its ability to bypass a T-T dimer and an 8-oxoG lesion efficiently and a T-T dimer the DNA this does not the ability of two Ts in the dimer to base with As J. Mol. Biol. PubMed Scopus Google Scholar, D. 1995; PubMed Scopus Google Scholar, J. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). 8-oxoG in T and has the correct geometry to base with A, 8-oxoG in the base pairs with C M. S. M. Johnson F. Biochemistry. 1991; PubMed Scopus (306) Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T. Biochemistry. PubMed Scopus Google Scholar, S. M. S. H. H. 1991; PubMed Scopus Google Scholar). The template is distorted in the of the lesion in the base M. S. M. Johnson F. Biochemistry. 1991; PubMed Scopus (306) Google Scholar, A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T. Biochemistry. PubMed Scopus Google Scholar, S. M. S. H. H. 1991; PubMed Scopus Google Scholar). yeast and human Polη incorporate As opposite the two Ts of the T-T dimer with the same efficiency and accuracy as opposite undamaged Ts M.T. Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3094-3099PubMed Google Scholar, R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In contrast with eukaryotic polymerases α, δ, and ε, which all bypass 8-oxoG by an A opposite the lesion, Polη bypasses 8-oxoG by inserting predominantly a C opposite the lesion (18Haracska L., Yu, S.-L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (306) Google Scholar). These and other (19Haracska L. Prakash S. Prakash L. Mol. Cell. Biol. 2000; 20: 8001-8007Crossref PubMed Scopus (118) Google have suggested that Polη is to geometric distortions upon DNA by these lesions. we examine the ability of Polη to bypass an AP site. abasic site is a prototypical noninstructional DNA lesion. have indicated that DNA containing an A opposite the AP site all of DNA, and the A and the abasic residue the R. B. Goodman M.F. M. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar, J. Mol. Biol. 1990; PubMed Scopus Google Scholar). The A is well in the as if with T, and the of the AP site is the same as that of the base R. B. Goodman M.F. M. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar, J. Mol. Biol. 1990; PubMed Scopus Google Scholar). low a G opposite the AP site is also predominantly J. Mol. Biol. 1990; PubMed Scopus Google Scholar). However, a is opposite the AP site, both the and the abasic are and the J. Mol. Biol. 1990; PubMed Scopus Google Scholar). DNA polymerases an A opposite the AP site 1991; 13: PubMed Scopus Google Scholar), the geometry of an A opposite an AP site resembles an base As from steady-state kinetic analyses of nucleotide insertion and both yeast and human Polη incorporate nucleotides opposite the AP site very and they are also highly inefficient in extension of the that Polη requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation. In the of either of these template either the or the DNA substrate a that is not to nucleotide incorporation. a in dNTP to the in the in for dNTP in both the incorporation opposite an AP site and the extension from bases opposite the AP site. the results here for a of Polη in AP they not the that with the bypass ability of this In the DNA synthesis efficiency of the and the efficiency is in for M. M. Woodgate R. Goodman M.F. Nature. 2000; PubMed Scopus Google Scholar). for AP bypass by nucleotide incorporation opposite the lesion by