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• W2022265644 abstract "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 relative electrophoretic mobility on 20% polyacrylamide gels (Fig.1 B, lanes 1–4). To facilitate bypass, we used high yPolη as well as high dNTP concentrations, which on the undamaged G template resulted in 100% synthesis to the end of the template DNA (Fig. 1 B, lane 5). Even under these forcing conditions, yPolη carried out almost no AP bypass, and only nucleotide incorporation opposite the AP site without further extension was observed (Fig. 1 B, lane 6). In the presence of all four dNTPs, yPolη inserted primarily a G residue (95%) across from the AP site (Fig. 1 B, lane 6). With only a single nucleotide present besides a G residue, yPolη also incorporated an A, and T was inserted very weakly opposite the AP site (Fig. 1 B, lanes 7–10). Next we measured the kinetics of nucleotide insertion and extension during DNA synthesis past the AP site. To determine the frequency of nucleotide incorporation by yPolη, we measured 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 Scholar) for all four incoming dNTPs opposite a template AP site. For purposes of comparison, the kinetic parameters opposite nondamaged template residues were measured as well. yPolη was incubated with the DNA substrate and with increasing concentrations of one of the four deoxynucleotides. The pattern of deoxynucleotide incorporation by yPolη opposite an AP site is shown in Fig.2 A. The Kmand Vmax parameters were determined and used to calculate the percentage of each nucleotide incorporated opposite the AP site (Table I). yPolη incorporated 59% G, 31% A, 7% T, and 3% C opposite the AP site. This analysis indicates that yPolη incorporates a G opposite the AP site with a 2-fold higher efficiency than A. Importantly, however, yPolη inserts a G opposite the AP site about 1,000-fold less efficiently than the insertion of G opposite C (Table I). The other nucleotides were inserted even less efficiently (Table I). The substantially lower efficiency of nucleotide incorporation opposite an AP site relative to a nondamaged template residue results from a 1,000–10,000-fold increase in the Km for dNTP (Table I).Table IKinetic parameters of insertion reactions catalyzed by yeast PolηSitedNTP addedKmVmaxVmax/KmInsertion 1-aCalculated by dividing the efficiency (Vmax/Km) of insertion for each dNTP by the sum of the insertion efficiencies of all four dNTPs.Relative efficiency 1-bCompares the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template base.μm%/min%Insertion opposite abasic site1-c0indicates an AP site.5′- - - -CTCdGTP51 ± 4.126 ± 1.80.51591 /878- - - - -GAG0T- -5′- - - -CTCdATP77 ± 4.521 ± 0.80.27311 /1988- - - - -GAG0T- -5′- - - -CTCdTTP194 ± 3712 ± 1.10.06271 /12790- - - - -GAG0T- -5′- - - -CTCdCTP248 ± 227.3 ± 0.80.02931 /66931- - - - -GAG0T- -Insertion opposite nondamaged site5′- - - -CTCdGTP0.058 ± 0.0226 ± 2.4448- - - - -GAGCT- -5′- - - -CTCdATP0.067 ± 0.0136 ± 1.8537- - - - -GAGTT- -5′- - - -CTCdTTP0.029 ± 0.0123 ± 1.5793- - - - -GAGAT- -5′- - - -CTCdCTP0.017 ± 0.00533 ± 1.91941- - - - -GAGGT- -1-a Calculated by dividing the efficiency (Vmax/Km) of insertion for each dNTP by the sum of the insertion efficiencies of all four dNTPs.1-b Compares the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template base.1-c 0indicates an AP site. Open table in a new tab For lesion bypass to occur, it is important that, after incorporating a nucleotide opposite the lesion, a polymerase extend the primer beyond 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 paired with an AP site were measured. Fig. 2 Bpresents the pattern of extension from G, A, T, or C when paired with an AP site. From the pattern of extension from these different 3′ termini, the Km and Vmaxparameters were obtained and used to calculate the efficiency (Vmax/Km) of extension. The ratio of extension opposite from the AP site was: G:A:T:C = 49:37:9:5 (Table II), which indicates that yPolη extends from a G or an A opposite the AP site about equally well. yPolη, however, extends from G or A quite inefficiently, as the efficiency (Vmax/Km) of extension in both cases was reduced by almost 1000-fold that from the opposite nondamaged complementary bases (Table II). The lower efficiency of extension from bases opposite an AP site relative to the extension from bases opposite a nondamaged residue was also because of a 500–2000-fold increase in the Km for dNTP (TableII). Hence, yPolη is very inefficient in inserting nucleotides across from an AP site as well as in extending from the nucleotide inserted.Table IIKinetic parameters of extension reactions catalyzed by yeast PolηSitedNTP addedKmVmaxVmax/KmExtension 2-aCalculated by dividing the efficiency (Vmax/Km) of each extension by the sum of the efficiencies of all four extensions.Relative efficiency 2-bCompares the efficiency of extension from a nucleotide placed opposite the template AP site to the efficiency of extension from the nucleotide opposite from the complementary template base.μm%/min%Extension from G, A, T, or C opposite abasic site2-c0indicates an AP site.5′- - - -TCGdATP85 ± 7.411 ± 0.60.129491 /906- - - - -AG0TC- -5′- - - -TCAdATP114 ± 2111 ± 1.60.096371 /885- - - - -AG0TC- -5′- - - -TCTdATP182 ± 374.5 ± 1.10.02591 /3480- - - - -AG0TC- -5′- - - -TCCdATP257 ± 463.3 ± 0.80.01351 /9769- - - - -AG0TC- -Extension from G, A, T, or C opposite nondamaged site5′- - - -TCGdATP0.18 ± 0.0421 ± 2.4117- - - - -AGCTC- - -5′- - - -TCAdATP0.21 ± 0.0218 ± 0.5385- - - - -AGTTC- -5′- - - -TCTdATP0.24 ± 0.0221 ± 2.687- - - - -AGATC- -5′- - - -TCCdATP0.15 ± 0.0419 ± 1.1127- - - - -AGGTC- -2-a Calculated by dividing the efficiency (Vmax/Km) of each extension by the sum of the efficiencies of all four extensions.2-b Compares the efficiency of extension from a nucleotide placed opposite the template AP site to the efficiency of extension from the nucleotide opposite from the complementary template base.2-c 0indicates an AP site. Open table in a new tab We also examined the ability of the human DNA polymerase η (hPolη) to bypass the AP site. Like yPolη, hPolη bypasses the AP site very inefficiently. hPolη also exhibits two stall sites, one right before the AP site and the other opposite the lesion, indicating that there is inhibition of deoxynucleotide insertion opposite the AP site as well as inhibition of extension from this lesion (data not shown). The extremely restricted ability of hPolη to bypass an AP site is further reflected in its steady-state Km andVmax kinetic values. The kinetics of insertion of a single deoxynucleotide opposite an AP site and the kinetics of addition of the next correct nucleotide to various 3′-primer termini situated across from the AP site were determined as a function of deoxynucleotide concentration. hPolη also inserts a G somewhat better than an A opposite the AP site. However, hPolη inserts these nucleotides opposite the AP site ∼103-fold less efficiently than opposite the nondamaged complementary base, because of a 600–2,500-fold increase in the Km for dNTP (TableIII). The order and the ratio of deoxynucleotide insertion opposite the AP site by hPolη were G:A:C:T ∼ 13:10:1.5:1 (Table III). hPolη also extends inefficiently from the nucleotide inserted opposite the AP site, and the order and the frequency of extension from different 3′-terminal deoxynucleotides paired with the AP site were A:G:C:T ∼ 5:2:2:1 (Table IV). Thus, human Polη inserts G slightly better than A opposite the AP site, but it is 2.5-fold more efficient at extending from an A opposite the AP site than from a G opposite this lesion. The poor extension efficiency of hPolη is again because of a 500–1500-fold increase in the Km for dNTP (Table IV).Table IIIKinetic parameters of insertion reactions catalyzed by human PolηSitedNTP addedKmVmaxVmax/KmInsertion 3-aCalculated by dividing the efficiency (Vmax/Km) of insertion for each dNTP by the sum of the insertion efficiencies of all four dNTPs.Relative efficiency3-bCompares the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template base.μm%/min%Insertion opposite abasic site3-c0indicates an AP site.5′- - - -CTCdGTP33 ± 6.719 ± 10.54511 /825- - - - -GAG0T- -5′- - - -CTCdATP39 ± 4.516 ± 1.30.41391 /1414- - - - -GAG0T- -5′- - - -CTCdTTP253 ± 2311 ± 0.80.04341 /3953- - - - -GAG0T- -5′- - - -CTCdCTP106 ± 126.3 ± 1.60.05961 /4338- - - - -GAG0T- -Insertion opposite nondamaged site5′- - - -CTCdGTP0.056 ± 0.00825 ± 2.15446- - - - -GAGCT- -5′- - - -CTCdATP0.062 ± 0.00736 ± 0.53580- - - - -GAGTT- -5′- - - -CTCdTTP0.1 ± 0.0217 ± 1.6170- - - - -GAGAT- -5′- - - -CTCdCTP0.082 ± 0.00321 ± 0.8256- - - - -GAGGT- -3-a Calculated by dividing the efficiency (Vmax/Km) of insertion for each dNTP by the sum of the insertion efficiencies of all four dNTPs.3-b Compares the efficiency of dNTP insertion opposite the template AP site to the efficiency of dNTP insertion opposite the complementary template base.3-c 0indicates an AP site. Open table in a new tab Table IVKinetic parameters of extension reactions catalyzed by human PolηSitedNTP addedKmVmaxVmax/KmExtension 4-aCalculated by dividing the efficiency (Vmax/Km) of each extension by the sum of the efficiencies of all four extensions.Relative efficiency 4-bCompares the efficiency of extension from a nucleotide placed opposite the template AP site to the efficiency of extension from the nucleotide opposite the complementary template base.μm%/min%Extension from G, A, T, or C opposite abasic site4-c0indicates an AP site.5′- - - -ATGdTTP124 ± 1313 ± 0.340.1181 /956- - - - -TA0AAT- -5′- - - -ATAdTTP104 ± 931 ± 1.70.29521 /582- - - - -TA0AAT- -5′- - - -ATTdTTP278 ± 3716 ± 2.30.057101 /3035- - - - -TA0AAT- -5′- - - -ATCdTTP146 ± 1917 ± 0.80.116201 /1896- - - - -TA0AAT- -Extension from G, A, T, or C opposite nondamaged site5′- - - -ATGdTTP0.23 ± 0.0422 ± 2.295.6- - - - -TACAAT- -5′- - - -ATAdTTP0.16 ± 0.0327 ± 0.5169- - - - -TATAAT- -5′- - - -ATTdTTP0.19 ± 0.0233 ± 1.6173- - - - -TAAAAT- -5′- - - -ATCdTTP0.1 ± 0.0122 ± 2.3220- - - - -TAGAAT- -4-a Calculated by dividing the efficiency (Vmax/Km) of each extension by the sum of the efficiencies of all four extensions.4-b Compares the efficiency of extension from a nucleotide placed opposite the template AP site to the efficiency of extension from the nucleotide opposite the complementary template base.4-c 0indicates an AP site. Open table in a new tab Polη is unique among eukaryotic DNA polymerases in its ability to bypass a cis-syn T-T dimer and an 8-oxoG lesion efficiently and accurately. Although a cis-syn T-T dimer disrupts the DNA helix, this distortion does not affect the ability of two Ts in the dimer to base pair with As (26Ciarrocchi G. Pedrini A.M. J. Mol. Biol. 1982; 155: 177-183Crossref PubMed Scopus (75) Google Scholar, 27Kim J.-K. Patel D. Choi B.-S. Photochem. Photobiol. 1995; 62: 44-50Crossref PubMed Scopus (201) Google Scholar, 28Husain I. Griffith J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2558-2562Crossref PubMed Scopus (137) Google Scholar). 8-oxoG in thesyn conformation mimics T and has the correct geometry to base pair with A, whereas 8-oxoG in the anti-conformation base pairs with C (29Kouchakdjian M. Bodepudi V. Shibutani S. Eisenberg M. Johnson F. Grollman A.P. Patel D.J. Biochemistry. 1991; 30: 1403-1412Crossref PubMed Scopus (306) Google Scholar, 30Lipscomb L.A. Peek M.E. Morningstar M.L. Verghis S.M. Miller E.M. Rich A. Essignman J.M. Williams L.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 719-723Crossref PubMed Scopus (222) Google Scholar, 31McAuley-Hecht K.E. Leanoard G.A. Gibson N.J. Thomson J.B. Watson W.P. Hunter W.N. Brown T. Biochemistry. 1994; 33: 10266-10270Crossref PubMed Scopus (223) Google Scholar, 32Oda Y. Uesugi S. Ikehara M. Nishimura S. Kawase Y. Ishikawa H. Inoue H. Ohtsuka E. Nucleic Acids Res. 1991; 19: 1407-1412Crossref PubMed Scopus (204) Google Scholar). The template strand, however, is significantly distorted in the vicinity of the lesion in the 8-oxoG·C base pair (29Kouchakdjian M. Bodepudi V. Shibutani S. Eisenberg M. Johnson F. Grollman A.P. Patel D.J. Biochemistry. 1991; 30: 1403-1412Crossref PubMed Scopus (306) Google Scholar, 30Lipscomb L.A. Peek M.E. Morningstar M.L. Verghis S.M. Miller E.M. Rich A. Essignman J.M. Williams L.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 719-723Crossref PubMed Scopus (222) Google Scholar, 31McAuley-Hecht K.E. Leanoard G.A. Gibson N.J. Thomson J.B. Watson W.P. Hunter W.N. Brown T. Biochemistry. 1994; 33: 10266-10270Crossref PubMed Scopus (223) Google Scholar, 32Oda Y. Uesugi S. Ikehara M. Nishimura S. Kawase Y. Ishikawa H. Inoue H. Ohtsuka E. Nucleic Acids Res. 1991; 19: 1407-1412Crossref PubMed Scopus (204) Google Scholar). Both 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 (12Washington M.T. Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3094-3099PubMed Google Scholar, 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). In contrast with eukaryotic replicative polymerases α, δ, and ε, which all bypass 8-oxoG by incorporating 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 observations (19Haracska L. Prakash S. Prakash L. Mol. Cell. Biol. 2000; 20: 8001-8007Crossref PubMed Scopus (118) Google Scholar) have suggested that Polη is refractory to geometric distortions conferred upon DNA by these lesions. Here we examine the ability of Polη to bypass an AP site. An abasic site is a prototypical noninstructional DNA lesion. NMR studies have indicated that DNA containing an A opposite the AP site retains all aspects of B-form DNA, and the unpaired A and the abasic residue lie inside the helix (33Cuniasse P. Sowers L.C. Eritja R. Kaplan B. Goodman M.F. Cognet J.A.H. LeBret M. Guschlbauer W. Fazakerley G.V. Nucleic Acids Res. 1987; 15: 8003-8022Crossref PubMed Scopus (106) Google Scholar, 34Kalnik M.W. Chang C.-N. Grollman A.P. Patel D.J. Biochemistry. 1988; 27: 924-931Crossref PubMed Scopus (105) Google Scholar, 35Cuniasse P. Fazakerley G.V. Guschlbauer W. Kaplan B.E. Sowers L.C. J. Mol. Biol. 1990; 213: 303-314Crossref PubMed Scopus (167) Google Scholar). The A is held well in the helix as if paired with T, and the melting temperature of the A· AP site is the same as that of the A·T base pair (33Cuniasse P. Sowers L.C. Eritja R. Kaplan B. Goodman M.F. Cognet J.A.H. LeBret M. Guschlbauer W. Fazakerley G.V. Nucleic Acids Res. 1987; 15: 8003-8022Crossref PubMed Scopus (106) Google Scholar, 34Kalnik M.W. Chang C.-N. Grollman A.P. Patel D.J. Biochemistry. 1988; 27: 924-931Crossref PubMed Scopus (105) Google Scholar, 35Cuniasse P. Fazakerley G.V. Guschlbauer W. Kaplan B.E. Sowers L.C. J. Mol. Biol. 1990; 213: 303-314Crossref PubMed Scopus (167) Google Scholar). At low temperatures, a G opposite the AP site is also predominantly intrahelical (35Cuniasse P. Fazakerley G.V. Guschlbauer W. Kaplan B.E. Sowers L.C. J. Mol. Biol. 1990; 213: 303-314Crossref PubMed Scopus (167) Google Scholar). However, when a pyrimidine is positioned opposite the AP site, both the pyrimidine and the abasic sugar are extrahelical, and the helix collapses (35Cuniasse P. Fazakerley G.V. Guschlbauer W. Kaplan B.E. Sowers L.C. J. Mol. Biol. 1990; 213: 303-314Crossref PubMed Scopus (167) Google Scholar). Many DNA polymerases insert an A opposite the AP site (36Strauss B.S. BioEssays. 1991; 13: 79-84Crossref PubMed Scopus (249) Google Scholar), presumably because the geometry of an A opposite an AP site closely resembles an A·T base pair. As revealed from steady-state kinetic analyses of nucleotide insertion and extension, both yeast and human Polη incorporate nucleotides opposite the AP site very inefficiently, and they are also highly inefficient in subsequent extension of the primer. This suggests that Polη requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation. In the absence of either of these template bases, either the enzyme or the enzyme-bound DNA substrate may adopt a conformation that is not conducive to nucleotide incorporation. Such a conformational alteration could then result in weaker dNTP binding to the enzyme-DNA complex resulting in the substantial increase in theKm for dNTP observed in both the incorporation opposite an AP site and the extension from bases opposite the AP site. Although the results reported here provide no compelling evidence for a role of Polη in AP bypass, they do not exclude the possibility that association with accessory factors modifies the damage bypass ability of this polymerase. In Escherichia coli, RecA stimulates the DNA synthesis efficiency of the UmuCD′ complex (PolV) 15,000-fold, and the increased efficiency is reflected mainly in theKm reduction for dNTPs (37Tang M. Pham P. Shen X. Taylor J.-S. O'Donnell M. Woodgate R. Goodman M.F. Nature. 2000; 404: 1014-1018Crossref PubMed Scopus (393) Google Scholar). By reducing theKm for dNTPs, accessory factors may facilitate AP bypass by promoting nucleotide incorporation opposite the lesion by Polη." @default.
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• W2022265644 title "Inefficient Bypass of an Abasic Site by DNA Polymerase η" @default.
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