Fidelity of Escherichia coli DNA Polymerase III Holoenzyme
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.27919
ISSN1083-351X
AutoresLinda B. Bloom, Xiluo Chen, Deborah Kuchnir Fygenson, Jennifer Turner, Mike O’Donnell, Myron F. Goodman,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of β, γ processivity and ε proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f inc = 5.6 × 10−6, 4.2 × 10−7, and 7 × 10−7, respectively. Elevated dGMP·G and dTMP·G misincorporation efficiencies of 3.2 × 10−5 and 5.8 × 10−4, attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the α holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP·G, dTMP·G, and dAMP·G mispairs. Both the β, γ complex and the downstream nucleotide have little effect on the fidelity of catalytic α subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931–27935 (accompanying paper)). The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of β, γ processivity and ε proofreading subunits are evaluated using a gel kinetic assay. Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f inc = 5.6 × 10−6, 4.2 × 10−7, and 7 × 10−7, respectively. Elevated dGMP·G and dTMP·G misincorporation efficiencies of 3.2 × 10−5 and 5.8 × 10−4, attributed to a "dNTP-stabilized" DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of misinserted nucleotides are excised by pol III holoenzyme in the absence of a next correct "rescue" nucleotide. As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide. Compared with pol III holoenzyme, the α holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP·G, dTMP·G, and dAMP·G mispairs. Both the β, γ complex and the downstream nucleotide have little effect on the fidelity of catalytic α subunit. An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the "Appendix" (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem. 272, 27931–27935 (accompanying paper)). The first in vitro measurement of DNA synthesis fidelity was carried out by Kornberg and co-workers in 1962 (1Trautner T.A. Swartz M.N. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1962; 48: 449-455Crossref PubMed Scopus (62) Google Scholar) to analyze the mutagenic behavior of 5-bromouracil. Pol I 1The abbreviations used are: pol I, E. coli DNA polymerase I; pol III, E. coli DNA polymerase III; pol III core, comprised of α (polymerase), ε (3′ → 5′ proofreading exonuclease), and θ; pol III holoenzyme, comprised of pol III core + β (sliding processivity clamp) + γ complex (clamp loading complex containing γ, γ′, δ, χ, ψ); α holoenzyme, comprised of α + β, γ complex; pol δ; eucaryotic DNA polymerase δ; p/t, primer/template; SSB, E. coli single-stranded DNA-binding protein; T (in boldface), refers to the template target site at which fidelity is measured and should not be confused with T which refers to a template thymine; SCH, single completed hit conditions referring to extension of a primer via interaction with a DNA polymerase at most once, followed by polymerase dissociation; MCH, multiple completed hit conditions referring to extension of a primer via multiple interactions with DNA polymerase. was found to misincorporate dGMP more readily opposite template bromouracil than opposite T, thus providing a biochemical basis for understanding bromouracil's ability to stimulate A·T → G·C transition mutations in Escherichia coli and bacteriophage T4 (2Freese E. Proc. Natl. Acad. Sci. U. S. A. 1959; 45: 622-633Crossref PubMed Google Scholar, 3Freese E. J. Mol. Biol. 1959; 1: 87-105Crossref Scopus (206) Google Scholar). The next 3 decades bore witness to a wide range of fidelity studies investigating the biochemical basis of spontaneous and base analog-induced mutagenesis. These studies focused primarily on elucidating the properties of DNA polymerases and proofreading 3′-exonucleases and on developing and implementing methods to measurein vitro and in vivo mutational spectra. A comprehensive review of the first 30 years of fidelity studies is contained in Refs. 4Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Crossref PubMed Scopus (620) Google Scholar and 5Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Crossref PubMed Scopus (404) Google Scholar. DNA polymerases by themselves synthesize DNA with relatively low processivity. However, polymerases can interact with groups of accessory proteins to carry out processive chromosomal replication. The first experiments to identify and characterize proteins that confer high processivity were performed by Alberts, Nossal, and co-workers (6Nossal N.G. Alberts B.M. Mathews C.K. Kutter E.M. Mosig G. Berget P.B. Bacteriophage T4. American Society for Microbiology, Washington, D. C.1983: 71-81Google Scholar,7Nossal N.G. Karam J.D. Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, D. C.1994: 43-53Google Scholar) using T4 bacteriophage. These studies were instrumental for the subsequent development of in vitro systems to study replication holoenzymes from E. coli (8Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6561-6569Abstract Full Text PDF PubMed Google Scholar, 9McHenry C.S. Annu. Rev. Biochem. 1988; 57: 519-550Crossref PubMed Scopus (111) Google Scholar, 10Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar), T7 bacteriophage (11Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar), and eucaryotes (12Chalberg M.D. Kelly T.J. Annu. Rev. Biochem. 1989; 58: 671-717Crossref PubMed Google Scholar, 13Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar). E. coli β subunit is required to allow pol III core to attain high processivity (8Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6561-6569Abstract Full Text PDF PubMed Google Scholar, 14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). X-ray diffraction data show that the β subunit is a doughnut-shaped dimer that can form a ring around DNA (15Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar) and functions as a sliding clamp that inhibits dissociation of pol III core from DNA during chain elongation (14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). The five-subunit γ complex is required to load β onto DNA (9McHenry C.S. Annu. Rev. Biochem. 1988; 57: 519-550Crossref PubMed Scopus (111) Google Scholar, 10Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). The processivity of pol III core alone is only 10–20 nucleotides (16Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar), while in the presence of β and γ complex it is increased to greater than 5000 nucleotides (8Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6561-6569Abstract Full Text PDF PubMed Google Scholar, 14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). Analysis of the specific effects of β, γ complex on fidelity is key to understanding the effect of processivity proteins on base substitution and frameshift mutations. It has been shown that mutations in T4 polymerase accessory proteins in vivo can cause both mutator and antimutator effects on the levels of spontaneous base substitution mutations (17Watanabe S.M. Goodman M.F. J. Virol. 1978; 25: 73-77Crossref PubMed Google Scholar). A measurement of T7 pol fidelity in anin vitro gap-filling assay suggests that processive synthesis, requiring the presence of thioredoxin, leads to an elevation in base substitutions and simple frameshift mutations in nonrepetitive sequences (18Kunkel T.A. Patel S.S. Johnson K.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6830-6834Crossref PubMed Scopus (92) Google Scholar). A recent study of eucaryotic pol δ suggests that nucleotide misincorporation efficiencies are increased in the presence of the proliferating cell nuclear antigen sliding clamp, a eucaryotic sliding clamp analogous to the E. coli β subunit (19Mozzherin D.Ju. McConnell M. Jasko M.V. Krayevsky A.A. Tan C.-K. Downey K.M. Fisher P.A. J. Biol. Chem. 1996; 271: 31711-31717Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this paper, we analyze the individual and combined effects of the β, γ complex and ε proofreading exonuclease on nucleotide misincorporation efficiencies using a generalized gel kinetic assay (20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). We compare fidelities at the same p/t DNA site for α subunit, α holoenzyme, pol III core, and pol III holoenzyme; α is the polymerase subunit of pol III core, which lacks proofreading activity. We also quantify a fundamental hallmark of proofreading, the dependence of pol III holoenzyme fidelity on the concentration of next correct dNTP. Fidelity measurements for enzymes with poor processivity and/or high fidelity, such as are reported here, require pushing the limits of the gel kinetic assay. In the "Appendix" (see accompanying paper (60Fygenson D.K. Goodman M.F. J. Biol. Chem. 1997; 272: 27931-27935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar)), we provide an analysis that extends the usefulness of the assay by allowing for multiple encounters between DNA polymerase and DNA. This important generalization facilitates measurements of high fidelities with or without proofreading or processivity proteins. E. coli DNA pol III proteins were purified as described: α, ε, and γ (21Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar); β (15Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar); δ and δ′ (22Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar); χ and ψ (23Xiao H. Crombie R. Dong Z. Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11773-11778Abstract Full Text PDF PubMed Google Scholar); and θ (24Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 19833-19841Abstract Full Text PDF PubMed Google Scholar). Subassemblies of γ complex and core were constituted as described (24Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 19833-19841Abstract Full Text PDF PubMed Google Scholar, 25Onrust R. Finkelstein J. Naktinis V. Turner J. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13348-13357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Enzyme reaction buffer contained 20 mm Tris-HCl, pH 7.5, 40 μg/ml bovine serum albumin, 5 mm dithiothreitol, 50 mm NaCl, and 8 mm MgCl2. T4 polynucleotide kinase was purchased from U. S. Biochemical Corp. or Amersham Corp. T4 DNA ligase was purchased from Promega. E. coli SSB was purchased from Pharmacia Biotech Inc. Two 30-mer primers, one 80-mer, and two 100-mer templates were used. The primers were annealed to the middle of their templates with equal lengths (25 or 35 nucleotides) of single-stranded DNA overhang on each side. Both 30-mer primers, two 40-mers, and three 50-mers, which were the 3′- and 5′-half of 80-mer and 100-mer templates, respectively, were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer. The 3′-half of a template was 5′-end-phosphorylated by ATP using T4 polynucleotide kinase. The two halves were then annealed to the 30-mer primer and ligated to form the full template. Primers and templates were gel-purified. The sequences of the 30-mer primer/80-mer template were as follows. The sequences of the 30-mer primer/100-mer templates were as follows. G is the target site where misincorporation frequencies are measured. The next nucleotide downstream from the target G is either C or A. dNTP substrates were purchased from Pharmacia Biotech Inc. [γ-32P]ATP (4500 Ci/mmol) was purchased from ICN Radiochemicals. The primer was 5′-end-labeled with32P using T4 polynucleotide kinase in enzyme reaction buffer at 37 °C for 60 min. p/t DNA was annealed in enzyme reaction buffer using a ratio of 1 primer to 1.2 templates by heating to 90 °C and gradually cooling to room temperature. The concentration of p/t DNA after annealing was 100 nm (primer terminus). p/t DNA and ATP were incubated at 37 °C with different combinations of accessory proteins (β, γ complex) and SSB in 20 μl of enzyme reaction buffer containing 4% glycerol for 4 min; then, 10 μl of enzyme reaction buffer containing 4% glycerol, four dNTPs, and pol III core was added to start the reaction. Reactions were run for 3 min at 37 °C and quenched by adding 60 μl of 20 mm EDTA in 95% formamide (F/E) to the reaction mixture. Final concentrations in the reactions were 5 nm p/t, 5 nm pol III core, 167 μm ATP, 133 μm dATP, 133 μm dCTP, 133 μm dGTP, 133 μmdTTP, and the accessory proteins, if present, were 20 nmγ complex, 80 nm β2, 320 nmSSB. Primer extension reactions were carried out using pol III core, pol III holoenzyme, α subunit, and α holoenzyme. All reactions were carried out in a similar way. Time courses were run prior to kinetics experiments to determine conditions for measuring incorporation opposite a target site. Three solutions were made for measurements using pol III core in the presence of β, γ complex and SSB. Solution A contained 15 nm p/t, 60 nm γ complex, 240 nmβ2, 960 nm SSB, and 4% glycerol in enzyme reaction buffer. Solution B consisted of enzyme reaction buffer containing 0.5 mm ATP and varied concentrations of the dNTP to be incorporated opposite target site. Solution C contained 15 nm of pol III core, 150 μm running start dATP, and 4% glycerol in enzyme reaction buffer. The reaction was performed as follows: 5 μl of solution A was first mixed with 5 μl of solution B and incubated at 37 °C for 4 min to allow the γ complex to load β sliding clamp onto the DNA; then 5 μl of solution C was added to the A + B mixture to initiate the primer extension reaction. The concentrations in the final 15-μl reaction mixture were 5 nm p/t, 5 nm pol III core, 20 nmγ complex, 80 nm β2, 320 nmSSB, 167 μm ATP, 50 μm dATP, and varied concentrations of the dNTP to be incorporated opposite the target site. Reactions were run at 37 °C for 6 min for misincorporation opposite a target site. This constituted a multiple completed hit (MCH) condition in which about 50% of the primers were used (see the "Appendix" (60Fygenson D.K. Goodman M.F. J. Biol. Chem. 1997; 272: 27931-27935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar)). For correct incorporation opposite the target site, 3 nm pol III core was present in solution C to give 1 nm in the final reaction, and the reactions were run for 1 min at 37 °C, creating single completed hit (SCH) conditions, in which about 10% of the primers were utilized. Reactions were quenched by adding 30 μl of F/E to the reaction mixture. The samples were heated to 100 °C for 6 min, placed on ice for 3 min, then loaded on a 16% polyacrylamide denaturing gel. The gel was run at 2000 V for 3 h to separate reaction products. Primer extension reactions by α subunit in the presence of β, γ complex and SSB were carried out similarly using 8 nm α subunit instead of pol III core polymerase in 6 min reactions for misincorporation (MCH conditions, 50% of primers used) and 2 nm α subunit in 1 min reactions for correct incorporation opposite the target site (SCH conditions, 10% of primers used). Primer extension by α subunit or pol III core without accessory proteins were done with 8 nm α subunit or 5 nm of pol III core for misincorporation and 1 nm α subunit or 0.5 nm of pol III core for correct incorporation opposite the target. Also, ATP was omitted from solution C, and the incubation of the A + B mixture was reduced to 1 min. Reactions by α subunit and by pol III core were run for 30 min for misincorporation (MCH condition) and 1 min for correct incorporation (SCH condition). 30-Min reactions utilized about 90% of the primers, while 1-min reactions utilized 10% of the primers. Experiments were identical to primer extension reactions with core holoenzyme, except that the concentration of rescue nucleotide, dCTP, was 3-fold greater in solution C than its final concentration in the assay. Misincorporation and correct incorporation reactions were both carried out with 5 nm pol III core and were run for 6 min (MCH condition, 50% of primers used). Misincorporation of the running start nucleotide, dAMP, opposite the target site and beyond was not detected for concentrations up to 100 μm dATP, in the absence of other nucleotides. A gel fidelity analysis, outlined in Fig.1, was used to determine the kinetics of incorporation of each of the four dNTPs opposite the target site as a function of dNTP concentration (20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar, 27Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar). Integrated polyacrylamide gel band intensities were measured on a PhosphorImager using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The nucleotide incorporation rate opposite a target site, in either the presence or absence of proofreading exonuclease activity, can be obtained by measuring I T Σ/I T−1, where I T Σ is the integrated gel band intensities of primers extended to the target site and beyond, and I T−1 is the integrated gel band intensity of primers extended to the site just prior to the target site (20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar). A plot of the relative incorporation rate, I T Σ/I T−1 as a function of dNTP substrate concentration results in a rectangular hyperbola whose slope in the initial linear region is the apparentV max/K m . ApparentK m and relative V max values (20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar) were obtained using a least squares fit to the rectangular hyperbola. The relative V max value is equal to the maximum value of I T Σ/I T−1 . In reactions where misincorporation opposite the target site was relatively inefficient, plots of I T Σ/I T−1 versus dNTP concentration showed little or no curvature, and apparent V max/K m values were obtained by a least squares fit of the data to a straight line. Apparent V max/K m values that were obtained under MCH conditions were corrected to SCH conditions (see the "Appendix" (60Fygenson D.K. Goodman M.F. J. Biol. Chem. 1997; 272: 27931-27935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar)). The misincorporation efficiency,f inc, which is the inverse of the fidelity, is given by the ratio, finc=fidelity−1=(Vmax/Km)W(Vmax/Km)REquation 1 where the subscripts W and R refer to wrong and right incorporations, respectively. Measurement errors forV max/K m are ± 30% and for f inc are ± 40% (one S.D.). 2 nm 30-mer/100-mer DNA was preincubated for 3 min with 100 nm β2, 20 nm γ complex, 300 nm SSB in the presence of 1 mm ATP, and 0.4 mm dGTP, 0.4 mmdTTP, which are the nucleotides at the 3′-end of the primer. 10 nm pol III core was then added to the mixture to allow formation of pol III holoenzyme on the p/t DNA. Because dATP was not present, an idling reaction occurred in which dTMP and dGMP could be removed and then added back on to the 3′-end of the primer, preventing degradation of the 5′-32P-labeled primer. In a second tube, 500 nm unlabeled 30-mer/80-mer p/t DNA (trap DNA) was preincubated for 3 min with 4.5 μm β2, 1 μm γ complex, 10 μm SSB, 1 mmATP in the presence of 0.4 mm dGTP, 0.4 mmdTTP, and 0.6 mm dATP. The contents of the pol III holoenzyme (5 μl) and trap preincubation mixture (containing dATP) (10 μl) were then immediately combined, and four separate reactions were carried out in which 5 μl of dCTP of 1.6 mm, the nucleotide to be incorporated opposite the target G site, was added at delay times of 0, 10, 20, and 40 s. The reaction products were separated by polyacrylamide gel electrophoresis and the proportion of DNA extended beyond the target site was graphed as a function of delay time. A band at the T+2 site was caused by misincorporation at the target site and was not considered true extension, because its intensity was independent of whether dCTP was added (see Fig. 3 A). The same general procedure was used to measure pol III dissociation at the T−1 site for M13 DNA and a 30-mer/209-mer p/t DNA, where the 209-mer template was a restriction fragment of M13 DNA. The procedure was modified for the full-length circular M13 DNA as follows: an unlabeled primer (30-mer) was annealed to the M13 DNA downstream of the 32P-labeled primer to form a 60-nt gap; SSB was not present in the reaction and trap solutions. The effectiveness of the unlabeled 30-mer/80-mer DNA trap was demonstrated by adding pol III core plus β, γ complex, dNTPs and ATP first to the trap and then mixing with the radioactive labeled p/t DNA, followed by the delayed addition of dCTP. The trap was found to be effective over the entire length of the experiment, because negligible DNA synthesis (<1%) occurred on the labeled p/t DNA (data not shown). Utilization of multiple p/t DNA molecules by pol III (i.e.cycling) was assayed by measuring the increase in the fraction of primer DNA extended as a function of time. Time course reactions were carried out with 20 nm 30-mer/100-mer using two concentrations of pol III holoenzyme (1 and 3 nm) in the presence of all four dNTPs (each at 500 μm). The procedure used was the same as in the primer extension reaction (see above). A gel kinetic assay (26Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar, 28Randall S.K. Eritja R. Kaplan B.E. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 6864-6870Abstract Full Text PDF PubMed Google Scholar) designed to measure DNA polymerase fidelity at arbitrary template sites in the absence of proofreading was recently generalized for use with polymerases that can proofread (20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The assay is described above (see "Experimental Procedures") and illustrated in Fig. 1. A more detailed description is contained in Refs. 20Creighton S. Goodman M.F. J. Biol. Chem. 1995; 270: 4759-4774Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and 27Creighton S. Bloom L.B. Goodman M.F. Methods Enzymol. 1995; 262: 232-256Crossref PubMed Scopus (226) Google Scholar and an important extension of it is given in the "Appendix" (60Fygenson D.K. Goodman M.F. J. Biol. Chem. 1997; 272: 27931-27935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Three experimental criteria must be satisfied to measure the fidelity of a holoenzyme using this assay: (i) the holoenzyme must be processive on the p/t DNA used in the assay; (ii) primer extension bands attributable to the core enzyme (without processivity proteins) must be negligible compared with holoenzyme-catalyzed extension bands; (iii) primer extension bands attributable to the holoenzyme must arise either from SCH, in which an enzyme encounters a given p/t DNA at most once and then dissociates, or alternatively from MCH, in which enzymes encounter and dissociate from a given p/t DNA multiple times; primer extension bands caused by incompleted hits, arising if an enzyme remains bound at the primer-3′ terminus when the reaction is terminated, must be negligible. All three criteria are satisfied in our system, as demonstrated in the next two subsections. A detailed mathematical analysis of the effects of SCH and MCH on the fidelity measurements is given in the "Appendix" (60Fygenson D.K. Goodman M.F. J. Biol. Chem. 1997; 272: 27931-27935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). On large DNA templates, such as φX and M13, pol III core polymerase typically incorporates between 10 and 20 nucleotides per binding event (16Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar), while pol III core plus β and γ complex (pol III holoenzyme) incorporates more than 5000 nucleotides per binding event (8Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6561-6569Abstract Full Text PDF PubMed Google Scholar, 14Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). This difference implies that the processivity proteins (β, γ complex) increase the time pol III core remains bound to p/t DNA by 2 to 3 orders of magnitude. We have recently shown that a DNA template as small as 80 nucleotides in length, annealed to a 30-mer primer, supports processive synthesis by pol III holoenzyme in the presence of SSB (29Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The SSB protein, although useful in eliminating secondary structure that can cause polymerase pausing, is generally not required for processive synthesis on long, closed circular DNAs such as bacteriophage φX or M13. In our 30-mer/100-mer or 30-mer/80-mer (29Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) constructs, however, SSB is required, perhaps to prevent the β clamp from sliding off the end of the template before it has had time to associate with a polymerase molecule. Fig. 2 illustrates the requirements for processive synthesis in our systems. Synthesis by pol III core is essentially nonprocessive on the 30-mer/100-mer p/t DNA (Fig. 2,lane 9). The addition of β or γ complex, alone or together, has little effect; the enzyme still fails to reach the end of the template strand and dissociation bands are evident at each template position (Fig. 2, lanes 6–8). SSB alone strongly inhibits core polymerase, even in the presence of either β or γ complex (Fig. 2, lanes 3–5). However, when β, γ complex and SSB are all present in the reaction, synthesis proceeds processively, reaching the end of the 100-mer template (Fig. 2, lane 2). Note that dissociation bands characteristic of the core enzyme (Fig. 2,lanes 6–9) are strongly attenuated during holoenzyme synthesis in the presence of SSB (Fig. 2, lane 2). The α subunit lacks the ε proofreading 3′-exonuclease of pol III core and is lower in polymerase activity than pol III core, but its requirements for processive synthesis are similar. On its own, it cannot replicate to the end of the 100-mer template (Fig. 2, lane 11) but in the presence of β, γ complex and SSB it becomes much more processive (Fig. 2, lane 10). Despite their high processivity, the pol III and α holoenzymes eventually dissociate from p/t DNA. We measured the lifetime of pol III holoenzyme on p/t DNA using three different templates: a 100-mer, a 209-nt restriction fragment of M13 DNA, and full-length circular M13 DNA. Pol III holoenzyme was loaded onto a labeled p/t DNA in the presence of dGTP and dTTP allowing the polymerase to idle. Next, dATP was added along with a large excess of unlabeled trap DNA, and the reaction was allowed to incubate for varying "delay" times before addition of dCTP allowed processive synthesis. For each p/t DNA, the fraction of extended primer decreased with increasing delay times, exhibiting a good fit to an exponential decay (Fig. 3 A). The lifetimes (t½ values) of the pol III-β-p/t complexes are 4.7 s for the 100-mer, 25 s for the 209-nt M13 fragment, and 46 s for the full-length M13 DNA (Fig. 3 A). Cycling of pol III holoenzyme is apparent from the level of primer utilization over time. In the extreme, if enzymes fail to dissociate from p/t DNA, the fraction of extended primers would saturate at the pol/DNA ratio. We measured an exponen
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