Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol η, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol η in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol η has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10−2 to 10−3. Also pol η has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol η's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors. The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol η, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol η in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol η has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10−2 to 10−3. Also pol η has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol η's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors. polymerase variant form of the cancer-prone genetic disorder xeroderma pigmentosum Mutations in the RAD30 gene of Saccharomyces cerevisiae confer moderate sensitivity to ultraviolet (UV) radiation, and genetic studies have indicated the involvement of this gene in error-free bypass of UV-damaged DNA (1McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 2Roush A.A. Suarez M. Friedberg E.C. Radman M. Siede W. Mol. Gen. Genet. 1998; 257: 686-692Crossref PubMed Scopus (127) Google Scholar, 3Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 15975-15977Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). RAD30encodes a DNA polymerase, pol η,1 that can efficiently and correctly bypass a cis-syn-thymine-thymine (T-T) dimer in template DNA by inserting two adenines opposite the two thymines of the dimer (4Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (690) Google Scholar). The Rad30 DNA polymerase activity is essential for resistance to UV radiation and for its role in error-free bypass (3Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 15975-15977Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Recently, the human homologue of pol η has been identified, and mutational alterations in this protein are responsible for the variant form of the cancer-prone genetic disorder xeroderma pigmentosum (XP-V) (5Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (665) Google Scholar, 6Masutani 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 (1134) Google Scholar). Most DNA polymerases misincorporate deoxynucleotides with very low frequencies, in part because they prefer to insert deoxynucleotides that form correct Watson-Crick base pairs and which do not distort the Watson-Crick geometry (7Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (611) Google Scholar). Thus, even though the capacity for base pairing recognition is retained in the cyclobutane pyrimidine dimer (8Kemmink J. Boelens R. Koning T. van der Marel G.A. van Boom J.H. Kaptein R. Nucleic Acids Res. 1987; 15: 4645-4653Crossref PubMed Scopus (100) Google Scholar,9Kim J.-K. Patel D. Choi B.-S. Photochem. Photobiol. 1995; 62: 44-50Crossref PubMed Scopus (196) Google Scholar), a T-T dimer is still a block to most DNA polymerases, including the eukaryotic replicative polymerase pol δ (4Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (690) Google Scholar), presumably because of the intolerance of their active site to DNA distortion caused by the dimer (10Ciarrocchi G. Pedrini A.M. J. Mol. Biol. 1982; 155: 177-183Crossref PubMed Scopus (75) Google Scholar, 11Wang C.-I. Taylor J.-S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9072-9076Crossref PubMed Scopus (97) Google Scholar). In contrast, the ability of pol η for efficient and correct bypass of a T-T dimer may derive from an unusual active site that is more tolerant of DNA distortions. In that case, pol η would be expected to have a low fidelity (see also the “Discussion”). Here we determine the fidelity of pol η by measuring the steady-state kinetics of correct and incorrect deoxynucleotide incorporation and also examine its processivity. We find that pol η is a low fidelity polymerase, and it incorporates only a few nucleotides before dissociating from the primer-template DNA substrate. This low processivity may restrict pol η synthesis to short patches, thereby minimizing the error frequency, and thus accounting for its role in error-free bypass of UV-damaged DNA. The following four (53-mer) oligodeoxynucleotides were used as templates, and they differ only in the underlined sequences: Template G, 5′-ATGCC TGCAC GAAGA GTTCC TAGTG CCTAC ACTGG AGTAC CGGAG CATCG TCG; Template A, 5′-ATGCC TGCAC GAAGA GTTCG CTATG CCTAC ACTGG AGTAC CGGAG CATCG TCG; Template T, 5′-ATGCC TGCAC GAAGA GTTCA GCTTG CCTAC ACTGG AGTAC CGGAG CATCG TCG; Template C, 5′-ATGCC TGCAC GAAGA GTTCT AGCTG CCTAC ACTGG AGTAC CGGAG CATCG TCG. The oligodeoxynucleotide primer, 5′-CGACG ATGCT CCGGT ACTCC AGTGT AGGCA, was 5′-32P-end-labeled using polynucleotide kinase (Roche Molecular Biochemicals) and γ-32PATP (Amersham Pharmacia Biotech). Labeled primer (0.5 μm) was then annealed to the various templates (0.8 μm each) in the presence of 50 mm Tris-HCl, pH 7.5, and 100 mmNaCl by heating to 90 °C for 2 min, followed by cooling to 25 °C over several hours. DNA polymerase η was expressed in yeast strain BJ5464 and purified as described previously (3Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 15975-15977Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 4Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (690) Google Scholar). Pol η (2 nm) was incubated with 5′-32P-end-labeled primer-template substrate (50 nm) and various concentrations of a single deoxynucleotide (dNTP) (0–500 μm) in 25 mm NaPO4, pH 7.0, 5 mm MgCl2, 5 mm dithiothreitol, 100 μg/ml bovine serum albumin, and 10% glycerol. Reactions were carried out at 25 °C for 2, 5, or 10 min and terminated by adding an equal volume of 150 mm EDTA containing 1.5% SDS. Quenched reactions were diluted 10 times in formamide loading buffer (80% deionized formamide, 10 mm EDTA, pH 8.0, 1 mg/ml xylene cyanol, and 1 mg/ml bromphenol blue), heated to 90 °C for 2 min, placed on ice, and products resolved on 10% polyacrylamide sequencing gels containing 5.5 m urea. Analysis of the deoxynucleotide incorporation assays was done as described previously (12Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (400) Google Scholar, 13Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (225) Google Scholar, 14Bloom L.B. Chen X. Fygenson D.K. Turner J. O'Donnell M. Goodman M.F. J. Biol. Chem. 1997; 272: 27919-27930Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Gel band intensities of the substrates and products were quantitated using a PhosphorImager and the ImageQuant software (Molecular Dynamics). For each concentration of dNTP, the observed rate of deoxynucleotide incorporation (V obs) was determined by dividing the relative amount of the extended product by the incubation time. The observed rate of deoxynucleotide incorporation was plotted as a function of dNTP concentration, and the data were fit by nonlinear regression using SigmaPlot 4.0 to the Michaelis-Menton equation describing a hyperbola as follows (Equation 1). Vobs=(Vmax×dNTP)/(Km+dNTP)(Eq. 1) Apparent K m and V maxsteady-state parameters for the incorporation of the correct and incorrect deoxynucleotides were obtained from the fit and used to calculate the frequency of deoxynucleotide misincorporation (f inc) using the following equation (Equation 2). finc=(Vmax/Km)incorrect/(Vmax/Km)correct(Eq. 2) Pol η (20 nm) was preincubated with the primer-template DNA substrate (20 nm) in 25 mm NaPO4, pH 7.0, 5 mmdithiothreitol, 100 μg/ml bovine serum albumin, and 10% glycerol for 20 min at 25 °C. Reactions were initiated by adding all four deoxynucleotides (200 μm each), 5 mmMgCl2, and excess sonicated herring sperm DNA (1 mg/ml) as a trap. To demonstrate the effectiveness of the trap, pol η was preincubuated with the trap DNA and the primer-template substrate before the addition of dNTPs and MgCl2. After various times, reactions were quenched and run on a 10% polyacrylamide gel as described for the deoxynucleotide incorporation assays. The processivity,P n, after each deoxynucleotide incorporation was calculated by a method derived from one previously described (15von Hippel P.H. Fairfield F.R. Dolejsi M.K. Ann. N. Y. Acad. Sci. 1994; 726: 118-131Crossref PubMed Scopus (72) Google Scholar). Briefly, gel band intensities of the deoxynucleotide incorporation products at the 240 s incubation time were quantitated using the PhosphorImager and ImageQuant software. First, for each deoxynucleotide addition n, the percentage of active polymerase molecules incorporating at least n deoxynucleotides is given by the following equation (Equation 3), %active polymerases atn=(In+In+1+…)×100%/(I1+I2+…+In+…)(Eq. 3) where I1 is the intensity of the band at position 1, I n is the intensity of the band at position n, and so on. For each deoxynucleotide incorporation n, processivity, P n, is the probability that the polymerase will incorporate the next deoxynucleotide rather than dissociating (15von Hippel P.H. Fairfield F.R. Dolejsi M.K. Ann. N. Y. Acad. Sci. 1994; 726: 118-131Crossref PubMed Scopus (72) Google Scholar) and is given by the following equation (Equation 4). Pn=%active polymerases atn+1/%active polymerases atn(Eq. 4) Applying Equation 3 to Equation 4 gives an expression forP n in terms of gel band intensities, and this equation was used to calculate the processivity as follows (Equation 5). Pn=(In+1+In+2+…)/(In+In+1+In+2+…)(Eq. 5) Fidelity is a measure of the frequency of incorporating a correctly base-paired versus an incorrectly base-paired deoxynucleotide (7Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (611) Google Scholar, 12Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (400) Google Scholar, 13Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (225) Google Scholar, 16Goodman M.F. Fygenson D.K. Genetics. 1998; 148: 1475-1482Crossref PubMed Google Scholar). To determine the frequency of misincorporation by pol η, we measured theV max and K m steady-state parameters for the incorporation of correct and incorrect deoxynucleotides opposite each template residue, using a standing-start assay, wherein the target template residue immediately follows the end of the primer (13Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (225) Google Scholar). Pol η (2 nm) was incubated with the primer-template DNA substrate (50 nm) and various concentrations of one of the four deoxynucleotides, after which reaction products were resolved by polyacrylamide gel electrophoresis and band intensities quantified. Fig. 1 A shows the deoxynucleotide incorporation pattern opposite a template T residue. The concentrations of dGTP, dTTP, and dCTP were varied from 0 to 200 μm, whereas the concentrations of dATP was varied from 0 to 10 μm. Under the reaction conditions, the kinetics of deoxynucleotide incorporation were linear with time. The kinetics of single deoxynucleotide incorporation opposite the template T residue are shown in Fig. 1 B. These data were fit to the Michaelis-Menton equation (Equation 1) and used to determine the apparent K m and V max values for each deoxynucleotide (Table I). The frequency of misincorporation, f inc, of G, T, and C opposite the template residue T was then calculated using Equation 2 (12Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (400) Google Scholar, 13Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (225) Google Scholar, 14Bloom L.B. Chen X. Fygenson D.K. Turner J. O'Donnell M. Goodman M.F. J. Biol. Chem. 1997; 272: 27919-27930Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 17Fersht A. Enzyme Structure and Mechanism. W. H. Freeman and Co., New York1984Google Scholar): f inc = (V max/K m)incorrect/(V max/K m)correct.As shown in Table I, for the incorporation of the incorrect deoxynucleotide G opposite the template T, theV max was 0.89 nm/min andK m was 99 μm, whereas for the incorporation of the correct deoxynucleotide A opposite the template T, the V max was 2.9 nm/min and theK m was 1.7 μm. Thusf inc for G opposite T is 5.3 × 10−3 (Table I). Similarly, f inc of T and C opposite the template T residue were 8.8 × 10−3 and 6.5 × 10−3, respectively (Table I).Table IFidelity of pol η by steady-state kineticsdNTPV maxK mV max/K mf incnm/minμmSubstrate S-1: template GdGTP0.48 ± 0.067180 ± 590.00273.8 × 10−4dATP0.20 ± 0.01092 ± 130.00223.1 × 10−4dTTP0.71 ± 0.02822 ± 2.50.0324.4 × 10−3dCTP3.1 ± 0.310.43 ± 0.247.2Substrate S-2: template AdGTP0.61 ± 0.0055100 ± 250.00612.6 × 10−3dATP0.38 ± 0.0087190 ± 100.00208.7 × 10−3dTTP3.0 ± 0.211.3 ± 0.312.3dCTP0.50 ± 0.01066 ± 3.20.00763.3 × 10−3Substrate S-3: template TdGTP0.89 ± 0.07499 ± 170.00905.3 × 10−3dATP2.9 ± 0.141.7 ± 0.251.7dTTP1.2 ± 0.01082 ± 180.0158.8 × 10−3dCTP0.52 ± 0.005548 ± 130.0116.5 × 10−3Substrate S-4: template CdGTP3.1 ± 0.365.0 ± 1.70.62dATP0.69 ± 0.16200 ± 780.00355.8 × 10−3dTTP0.15 ± 0.01475 ± 230.00203.2 × 10−3dCTP0.67 ± 0.05349 ± 130.0142.3 × 10−2 Open table in a new tab The V max and K msteady-state parameters for the misincorporation of deoxynucleotides opposite the other three template bases and the calculatedf inc values are also shown in Table I. The values for the f inc ranged from 3.1 × 10−4 for the misincorporation of A opposite a template G to 2.3 × 10−2 for the misincorporation of C opposite template C, and the average value for f inc was 6.0 × 10−3. Processivity is a measure of how many deoxynucleotides a DNA polymerase incorporates in a single DNA binding event (15von Hippel P.H. Fairfield F.R. Dolejsi M.K. Ann. N. Y. Acad. Sci. 1994; 726: 118-131Crossref PubMed Scopus (72) Google Scholar, 18Bambara R.A. Fay P.J. Mallaber L.M. Methods Enzymol. 1995; 262: 270-280Crossref PubMed Scopus (47) Google Scholar). To ensure that we were observing deoxynucleotide incorporation resulting from a single DNA binding event, we monitored DNA synthesis in the presence of an excess of nonradiolabeled, sonicated herring sperm DNA as a trap (Fig. 2 A). The reactions inlanes 1–6 (Fig. 2 A) were performed by first preincubating pol η with the DNA substrate for 20 min. Excess herring sperm DNA, MgCl2, and all four deoxynucleotides were then added to initiate the reaction. The excess herring sperm DNA is included to trap all pol η molecules that dissociated from the substrate ensuring that all DNA synthesis resulted from a single DNA binding event. The reactions in lanes 7–12 (Fig. 2 A) were performed by first preincubating pol η with the DNA substrate and the excess herring sperm DNA for 20 min followed by the addition of MgCl2 and deoxynucleotides. The lack of DNA synthesis in these lanes shows that the excess herring sperm DNA is sufficient to trap all pol η molecules. The processivity of a DNA polymerase is quantitatively expressed as the probability, P n, for each deoxynucleotide incorporation event n that the polymerase will move ahead to incorporate the next nucleotide n + 1 rather than dissociate from the DNA template (15von Hippel P.H. Fairfield F.R. Dolejsi M.K. Ann. N. Y. Acad. Sci. 1994; 726: 118-131Crossref PubMed Scopus (72) Google Scholar). First, the percentage of active polymerase molecules incorporating at least n deoxynucleotides was calculated using Equation 3 (see “Materials and Methods”). The percentage of active polymerases adding at least one deoxynucleotide was set as 100%, and the percentage of active polymerases decreased after each subsequent addition because of the dissociation of some fraction of polymerase molecules. For example, 93% added at least two deoxynucleotides, 88% added at least three deoxynucleotides, 80% added at least four deoxynucleotides, and so on (Fig. 2 B). On this DNA substrate, ∼50% of the pol η molecules incorporate at least six deoxynucleotides before dissociating from the DNA. Next, we calculated the processivity P n after each deoxynucleotide incorporation using the following equation (Equation 5) (15von Hippel P.H. Fairfield F.R. Dolejsi M.K. Ann. N. Y. Acad. Sci. 1994; 726: 118-131Crossref PubMed Scopus (72) Google Scholar): P n = (I n +1 + I n +2 + …)/(I n + I n +1 + I n +2 + …), where I n is the intensity of band n, I n +1 is the intensity of band n + 1, and so on. For example, of the polymerase molecules that incorporated one nucleotide, 93% incorporated at least one additional nucleotide. Thus,P 1 = 0.93. The values of P n ranged from 0.94 in the case of n = 2 deoxynucleotide additions to 0.40 in the case of n = 7 deoxynucleotide additions with an average value of 0.76 ± 0.20. Thus after each nucleotide incorporation, on average of the pol η molecules incorporate at least one additional while dissociate from the DNA substrate. Thus pol η DNA with low processivity. DNA polymerases incorporate deoxynucleotides with very low (7Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (611) Google Scholar, 12Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (400) Google Scholar, 13Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (225) Google Scholar, 16Goodman M.F. Fygenson D.K. Genetics. 1998; 148: 1475-1482Crossref PubMed Google Scholar). This fidelity has been to in part because of the intolerance of the active site to in DNA (7Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (611) Google Scholar). of several DNA polymerases have indicated that a in the is for the of the the primer and the and it an role in fidelity a J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). these are with the that the deoxynucleotide in the active site of the polymerase in a of the template Watson-Crick base pairing geometry the and the template base is for this essential to J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the geometry is not the will not be though the bases of a cyclobutane pyrimidine dimer can base with adenines (8Kemmink J. Boelens R. Koning T. van der Marel G.A. van Boom J.H. Kaptein R. Nucleic Acids Res. 1987; 15: 4645-4653Crossref PubMed Scopus (100) Google Scholar, J.-K. Patel D. Choi B.-S. Photochem. Photobiol. 1995; 62: 44-50Crossref PubMed Scopus (196) Google Scholar), the geometry of the dimer (10Ciarrocchi G. Pedrini A.M. J. Mol. Biol. 1982; 155: 177-183Crossref PubMed Scopus (75) Google Scholar, 11Wang C.-I. Taylor J.-S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9072-9076Crossref PubMed Scopus (97) Google Scholar) presumably polymerases, because they the distortion (7Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (611) Google Scholar). The ability of pol η to efficiently and correctly bypass would suggest that relative to other DNA polymerases, the active site of pol η has an of DNA distortions. The in inc, for DNA a steady-state kinetics assay, the error for DNA polymerase and DNA polymerase replicative DNA polymerases, were to from L.B. Chen X. Fygenson D.K. Turner J. O'Donnell M. Goodman M.F. J. Biol. Chem. 1997; 272: 27919-27930Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, S. Goodman M.F. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). an the synthesis of DNA in a from the gene of the eukaryotic replicative DNA polymerase pol δ was to have an error rate of about R.A. 1991; PubMed Scopus Google Scholar), whereas pol for DNA and pol in short base are and have an error rate of about 10−3 to 10−4 R.A. 1991; PubMed Scopus Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). using the steady-state kinetics assay, we find pol η to have an error rate of to 10−3. Thus, relative to these other DNA polymerases, pol η has a low fidelity, which may from an active site more tolerant of in DNA. would be of to a of pol η with the of other DNA polymerases to determine the of the flexibility of pol η's active site that gives it a for and a its low fidelity, pol η functions in error-free bypass of UV in yeast and of pol η the frequency of (1McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 3Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 15975-15977Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Mol. Biol. 1993; PubMed Scopus Google Scholar, S. J. 1993; PubMed Scopus Google Scholar), and as a from a of pol η has on as the rate of s is not in R. S. and L. Thus, is that pol η to the of in This because the DNA synthesis activity of pol η is Pol η may DNA to bypass thus to errors. the activity of pol η may be by the S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), which may have a role in pol η to DNA damage and in ensuring that the of pol η is to damage pol δ over after the damage has been by pol