Portal de Periódicos da CAPES

Influence of DNA Structure on DNA Polymerase β Active Site Function

2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês

10.1074/jbc.m404016200

1083-351X

William A. Beard, David D. Shock, Samuel H. Wilson,

RNA Interference and Gene Delivery

In the ternary substrate complex of DNA polymerase (pol) β, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol β on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (kcat/Km, dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C ∼ A/T > T/A ∼ C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since Kd(dGTP) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand. In the ternary substrate complex of DNA polymerase (pol) β, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol β on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (kcat/Km, dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C ∼ A/T > T/A ∼ C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since Kd(dGTP) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand. DNA polymerases select (bind and incorporate) a nucleoside triphosphate (dNTP) from a pool of structurally similar molecules to preserve Watson-Crick base pairing rules. DNA polymerase (pol) 1The abbreviations used are: pol, polymerase; THF, tetrahydrofuran. 1The abbreviations used are: pol, polymerase; THF, tetrahydrofuran. β is a model polymerase to study mechanisms utilized to assure efficient and faithful DNA synthesis. Its small size, lack of essential accessory proteins, and absence of a proofreading exonuclease have facilitated its biochemical, kinetic, and structural characterization. More importantly, it shares many general structural and mechanistic features exhibited by other DNA polymerases (1Beard W.A. Wilson S.H. Mutat. Res. 2000; 460: 231-244Google Scholar). DNA polymerase β contributes two important enzymatic activities during single-nucleotide base excision DNA repair. A deoxyribose phosphate lyase activity is associated with the amino-terminal 8-kDa lyase domain. This activity excises a deoxyribose phosphate intermediate during repair of abasic sites and generates a 5′-phosphate in a single-nucleotide gap. The nucleotidyl transferase activity of pol β is associated with the 31-kDa polymerase domain that fills the single-nucleotide gap. In addition, the polymerase activity of pol β is necessary for several alternate repair pathways that require longer gap-filling DNA synthesis (e.g. long patch base excision repair) (2Dianov G.L. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 1999; 274: 13741-13743Google Scholar, 3Horton J.K. Prasad R. Hou E. Wilson S.H. J. Biol. Chem. 2000; 275: 2211-2218Google Scholar). A general feature observed in the structures of all DNA polymerases that include a template overhang (i.e. single-stranded DNA) is that the trajectory of the template strand bends dramatically as it enters the polymerase active site (1Beard W.A. Wilson S.H. Mutat. Res. 2000; 460: 231-244Google Scholar). This serves at least two functions. First, it provides the polymerase the ability to assess whether geometrical constraints imposed by correct Watson-Crick hydrogen bonding occur, and second, it discourages the next templating base from prematurely entering the polymerase active site, which could result in the downstream template base coding for nucleotide insertion (deletion mutagenesis) (4Osheroff W.P. Beard W.A. Yin S. Wilson S.H. Kunkel T.A. J. Biol. Chem. 2000; 275: 28033-28038Google Scholar). To accurately replicate DNA, polymerases need to stabilize the coding templating base as well as the correct, but not incorrect, incoming nucleotide (5Beard W.A. Wilson S.H. Chem. Biol. 1998; 5: R7-R13Google Scholar). This is achieved through a series of protein- and substrate-induced conformational changes that result in a dNTP-binding pocket formed by the templating base, DNA duplex terminus, and enzyme. In the absence of an incoming nucleotide, the carboxyl-terminal N-subdomain 2A functionally based nomenclature is employed for pol β to avoid confusion generated by the architectural nomenclature that compares the polymerase domain to a handlike structure (13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). The N-subdomain contributes important interactions with the Nascent base pair and is equivalent to the fingers subdomain of right-handed DNA polymerases. 2A functionally based nomenclature is employed for pol β to avoid confusion generated by the architectural nomenclature that compares the polymerase domain to a handlike structure (13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). The N-subdomain contributes important interactions with the Nascent base pair and is equivalent to the fingers subdomain of right-handed DNA polymerases. (residues 262–335) of pol β is in an open conformation so that key polymerase side chains do not interact with the templating nucleotide. Upon binding the correct nucleotide, the N-subdomain closes on the nascent base pair (templating and incoming nucleotides), creating several key interactions with the enzyme (6Beard W.A. Wilson S.H. Structure. 2003; 11: 489-496Google Scholar). Accordingly, the constraints imposed by the dNTP-binding pocket are determined by DNA sequence (i.e. structure) as well as the conformational fluctuations that occur in response to enzyme and substrate binding. In the pol β closed ternary substrate complex, the nascent base pair is sandwiched between the duplex DNA terminus and α-helix N (Fig. 1). Lys280 and Asp276 of α-helix N contribute van der Waals interactions with the templating and incoming nucleotide bases, respectively, whereas Asn279 and Arg283 contribute DNA minor groove interactions. Structure-based site-directed mutagenesis of these residues has identified interactions that contribute to efficient DNA synthesis (7Beard W.A. Osheroff W.P. Prasad R. Sawaya M.R. Jaju M. Wood T.G. Kraut J. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12141-12144Google Scholar, 8Ahn J. Werneburg B.G. Tsai M.D. Biochemistry. 1997; 36: 1100-1107Google Scholar, 9Kraynov V.S. Werneburg B.G. Zhong X.J. Lee H. Ahn J.W. Tsai M.D. Biochem. J. 1997; 323: 103-111Google Scholar, 10Kraynov V.S. Showalter A.K. Liu J. Zhong X. Tsai M.-D. Biochemistry. 2000; 39: 16008-16015Google Scholar, 11Vande Berg B.J. Beard W.A. Wilson S.H. J. Biol. Chem. 2001; 276: 3408-3416Google Scholar, 12Skandalis A. Loeb L.A. Nucleic Acids Res. 2001; 29: 2418-2426Google Scholar, 13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). The specific contribution provided by these interactions appears to be dependent on the identity of the base pair that is formed. For example, decreasing the stacking interactions of residue 280 with the templating base by site-directed mutagenesis resulted in a more dramatic loss in binding affinity for incoming complementary pyrimidines than purines, indicating that the energetic contributions of specific side chain interactions are strongly dependent on the specific insertion (13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). Since the terminal base pair of the growing DNA duplex forms part of the binding pocket of the incoming nucleotide, we now examine the kinetic consequences of modifying the DNA structure at or near the polymerase active site. The structure of the dNTP-binding pocket was altered by varying the DNA sequence or introducing DNA mismatches (i.e. base substitution intermediates), "extra" nucleotides (i.e. frameshift intermediates), or abasic sites in the DNA duplex upstream of the polymerase active site. Materials—Poly(dA), p(dT)20, ultrapure deoxynucleoside triphosphates, [γ-32P]ATP, and MicroSpin G-25 columns were from Amersham Biosciences. [α-32P]TTP was from PerkinElmer Life Sciences, and DE81 filters were from Whatman. Protein Purification—Human DNA polymerase β was purified as described previously (14Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Google Scholar). Enzyme concentration was determined by Coomassie dye binding using purified pol β as a standard (15Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). The concentration of purified pol β was determined by total amino acid analysis. DNA Preparation—The sequence, structure, and nomenclature of the DNA substrates used in this study are illustrated in Fig. 2. An oligonucleotide DNA substrate containing a single-nucleotide gap at position 16 was prepared by annealing three gel-purified oligonucleotides (Oligos Etc., Wilsonville, OR, or Integrated DNA Technologies, Coralville, IA). Each oligonucleotide was suspended in 10 mm Tris-HCl, pH 7.4, and 1 mm EDTA, and the concentration was determined from their UV absorbance at 260 nm. The annealing reactions were carried out by incubating a solution of 10 μm primer with 12 μm of downstream and template oligonucleotides at 90–100 °C for 3 min followed by 30 min at 65 °C and then slow cooling to room temperature. Kinetic Assays—Steady-state kinetic parameters for single-nucleotide gap-filling reactions were determined by initial velocity measurements as described previously (13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). Unless noted otherwise, enzyme activities were determined using a standard reaction mixture (50 μl) containing 50 mm Tris-HCl, pH 7.4, 100 mm KCl, 5 mm MgCl2, and 200 nm single-nucleotide gapped DNA. In some instances requiring high dNTP concentrations (e.g. misinsertion reactions), the MgCl2 concentration was increased to assure that there was at least 5 mm free Mg2+ in the reaction mixture. Enzyme concentrations and reaction time intervals were chosen so that substrate depletion or product inhibition did not influence initial velocity measurements. Reactions were stopped with 20 μl of 0.5 m EDTA and mixed with an equal volume of formamide dye, and the products were separated on 12% denaturing polyacrylamide gels. The dried gels were analyzed using a PhosphorImager (Amersham Biosciences) to quantify product formation. Equilibrium Binding Constants for Gapped Heteropolymeric DNA Substrates—The equilibrium dissociation constants (i.e. Kd) for the binding of heteropolymeric DNA gapped substrates were determined by inhibition of pol β activity on a homopolymeric DNA as described previously (16Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Google Scholar). Enzyme activities were typically determined using a standard reaction mixture (50 μl) containing 50 mm Tris-HCl, pH 7.4, 100 mm KCl, 5 mm MnCl2, 30 μm [α-32P]dTTP, 30 or 300 nm poly(dA)-poly(dT)20 (Km; expressed as 3′-OH primer termini), and varying concentrations of competitor heteropolymeric DNA. Radioactive dTTP incorporation was not typically observed on the competitor DNA substrate because the templating base is not adenine. Reactions were initiated by the addition of 15 nm pol β, incubated at room temperature for 10 min, and stopped by the addition of 20 μl of 0.5 m EDTA. Quenched reaction mixtures were spotted on Whatman DE-81 filter disks and dried. Unincorporated [α-32P]dTTP was removed, and filters were counted as before (17Beard W.A. Wilson S.H. Biochemistry. 1993; 32: 9745-9753Google Scholar). Data were fitted to Equation 1 for competitive inhibition by nonlinear regression methods. kobs=(kcat×S)(S+Km(1+CKd)) Eq.1 The Michaelis constant, Km, and kcat for the homopolymeric DNA substrate (S) were determined in the absence of heteropolymeric competitor DNA (C). When competitor DNA binds tightly (Kd < polymerase concentration, E), an alternate form of Equation 1 (Equation 2) is necessary to account for the depletion of free inhibitor and enzyme as the EC complex is formed (18Kuzmic P. Hill C. Kirtley M.P. Janc J.W. Anal. Biochem. 2003; 319: 272-279Google Scholar). νobs=ν0[E−C−Ki, app+(E−C−Ki,app)2+4EKi, app2E] Eq.2 For a competitor inhibitor, the apparent inhibitor constant is given by Equation 3. Ki, app=Kd(1+SKm) Eq.3 Influence of the Identity of the DNA Duplex Terminus on Catalytic Efficiency—Steady-state kinetic analysis previously revealed a strong dependence of catalytic efficiency (kcat/Km)on the identity of the primer terminus for single-nucleotide gap filling by pol β (19Vaisman A. Warren M.W. Chaney S.G. J. Biol. Chem. 2001; 276: 18999-19005Google Scholar). That study examined the efficiency of dCTP insertion into a single-nucleotide gapped substrate with a templating guanine and systematically altered the primer terminus or downstream sequence to characterize the influence of DNA sequence on insertion efficiency. The downstream DNA sequence did not significantly alter the catalytic efficiency of nucleotide insertion. We have reexamined the influence of the identity of the primer terminus on insertion efficiency in a different DNA sequence context and with a different incoming nucleotide (i.e. dGTP rather than dCTP). The results are tabulated in Table I and indicate that the efficiency of dGTP insertion is strongly dependent on the identity of the matched (i.e. Watson-Crick base pair) primer terminus. As observed previously (19Vaisman A. Warren M.W. Chaney S.G. J. Biol. Chem. 2001; 276: 18999-19005Google Scholar), primer termini with a pyrimidine are extended more efficiently than those with a purine situated at the 3′ terminus (DNA terminus: template/primer, G/C ∼ A/T > T/A ∼ C/G).Table IKinetics of mispair extension by human DNA polymerase β Values represent the mean (S.E.) of at least two independent determinations. Assays were performed as outlined under "Experimental Procedures." The sequence of the single-nucleotide DNA substrate is illustrated in Figure 2A. Except for the G/G terminus, dGTP insertion opposite a templating cytosine was followed. For G/G, the kinetics of dATP insertion was determined (i.e. templating T). The data are tabulated in order of decreasing catalytic efficiencies relative to a matched terminus. The template base of the terminal mismatch is used as the reference nucleotide for the matched terminus.(X/Y)n- 1:ZnaIdentity of the template (X) primer (Y) terminus adjacent to the templating base (Z) at position nkcatKmkcat/KmfextbAs calculated from the ratio of catalytic efficiencies for correct insertion on a mismatched/matched primer terminus. Since Kd(matched) ∼ Kd(mismatched), fext ∼ fmin010-2s-1μm10-4μm-1s-110-4(A/T):C16 (2)0.5 (0.01)3200 (400)(A/C):C2.33 (0.08)21 (2)11 (1)34 (5)(A/A):C0.035 (0.002)4.9 (1.5)0.7 (0.2)2.2 (0.7)(A/G):C0.041 (0.001)63 (16)0.065 (0.017)0.2 (0.059)(C/G):C5.5 (0.3)1.4 (0.2)400 (60)(C/T):C1.74 (0.06)260 (20)0.67 (0.06)17 (3)(C/A):C0.28 (0.07)160 (20)0.18 (0.05)4 (1)(C/C):C0.283 (0.009)400 (40)0.071 (0.007)1.8 (0.3)(G/C):C34 (3)0.34 (0.06)10000 (2000)(G/T):C10.1 (0.6)20 (3)50 (6)50 (10)(G/A):C0.22 (0.04)47 (4)0.47 (0.09)0.47 (0.1)(G/G):CNDcND, not detectedNDNDND(G/G):T0.0123 (0.0009)8 (2)0.15 (0.04)0.19 (0.07)dCalculated from the corresponding efficiency for dATP insertion with the (G/C):T gapped DNA substrate(T/A):C7 (1)1.2 (0.2)600 (100)(T/G):C7.7 (0.4)42 (16)18 (7)300 (100)(T/C):C7.7 (0.1)89 (3)8.6 (0.3)140 (20)(T/T):C0.31 (0.03)23 (2)1.3 (0.2)22 (5)a Identity of the template (X) primer (Y) terminus adjacent to the templating base (Z) at position nb As calculated from the ratio of catalytic efficiencies for correct insertion on a mismatched/matched primer terminus. Since Kd(matched) ∼ Kd(mismatched), fext ∼ fmin0c ND, not detectedd Calculated from the corresponding efficiency for dATP insertion with the (G/C):T gapped DNA substrate Open table in a new tab Kinetics of Mispair Extension—Human DNA polymerase β produces base substitution errors at a frequency of 1.1 × 10-4 (T/dGTP) to 2.3 × 10-7 (C/dCTP) (Table II). For a base substitution error to become a mutation, the mispair must be extended. This is a kinetically challenging event for all DNA polymerases, most likely due to the aberrant nucleotide-binding pocket generated by the mispair. The kinetic analysis of mispair extension is more complex than that to determine the fidelity of DNA synthesis (20Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar, 21Creighton S. Huang M.M. Cai H. Arnheim N. Goodman M.F. J. Biol. Chem. 1992; 267: 2633-2639Google Scholar, 22Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Google Scholar). The expression to determine relative mismatch extension efficiency is given by Equation 4 (20Mendelman L.V. Petruska J. Goodman M.F. J. Biol. Chem. 1990; 265: 2338-2346Google Scholar). fext=kcat,wkcat,r×Kd,rKd,w×(Km,r+Pr[dNTP])(Km,w+Pw[dNTP]) Eq.4 This expression is the relative rate of adding a correct nucleotide (dNTP) onto a wrong (w, mismatched) and right (r, matched) primer termini at equal concentrations under steady-state conditions. The parameters Kd, Km, and P refer to the equilibrium DNA binding affinity, dNTP Michaelis constant, and processivity for correct insertion on a matched or mismatched primer terminus. Processivity is defined as the ratio of rate constants describing nucleotide insertion and DNA dissociation (i.e. kpol/koff). Thus, the relative efficiency for extending a mismatch is dependent on the correct nucleotide concentration with maximum discrimination occurring at infinitely low dNTP concentrations. This intrinsic mismatch extension efficiency is referred to as (fmin0)(Equation 5). fmin0=[(kcat/Km)w(kcat/Km)r]×Kd,rKd,w Eq.5 If the polymerase binds DNA with matched and mismatched primer termini with equal affinities, then (fmin0)is simply the ratio of catalytic efficiencies for insertion on a mismatched relative to that on a matched terminus.Table IIKinetics of misinsertion by human DNA polymerase β Values represent the mean (S.E.) of at least two independent determinations. Assays were performed as outlined under "Experimental Procedures." The sequence of the single-nucleotide DNA substrate is illustrated in Fig. 2A. The identity of the primer terminus is G/C (X/Y). The data for each templating base (Z) are tabulated in order of decreasing catalytic efficiencies.Z/dNTPkcatKm(dNTP)kcat/KmFidelityaFidelity = (kcat/Km)correct/(kcat/Km)incorrect10-2 s-1μm10-4 μm-1 s-1A/dTTPbData taken from Ref. 1345 (3)1.5 (0.1)3000 (300)1A/dGTP0.7 (0.4)425 (75)0.165 (0.098)18,180 (10,950)A/dCTP0.45 (0.1)795 (135)0.057 (0.016)52,630 (15,680)A/dATP0.0116 (0.0006)387 (3)0.003 (0.0002)1,000,000 (120,200)C/dGTP34 (3)0.34 (0.06)10000 (2000)1C/dATP0.270 (0.07)235 (34)0.115 (0.034)86,960 (31,040)C/dTTP0.094 (0.002)915 (25)0.01 (0.004)1,000,000 (447,200)C/dCTP0.02 (0.01)852 (548)0.0023 (0.0019)4,347,800 (3,695,400)G/dCTPbData taken from Ref. 1330 (3)0.4 (0.1)7500 (2019)1G/dTTPbData taken from Ref. 130.58 (0.25)490 (60)0.118 (0.053)63,560 (33,280)G/dATPbData taken from Ref. 130.14 (0.01)450 (50)0.031 (0.004)241,900 (72,200)G/dGTP0.0029 (0.0003)110 (12)0.0026 (0.0004)2,885,000 (894,400)T/dATPbData taken from Ref. 1369 (1)0.9 (0.2)7667 (1707)1T/dGTPbData taken from Ref. 133.76 (0.89)430 (50)0.87 (0.23)8813 (3061)T/dCTP1.10 (0.13)730 (13)0.151 (0.018)50,770 (12,820)T/dTTP0.032 (0.002)640 (40)0.0050 (0.0004)1,533,000 (362,800)a Fidelity = (kcat/Km)correct/(kcat/Km)incorrectb Data taken from Ref. 13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar Open table in a new tab DNA Binding Affinity—We have previously utilized a simple competition assay to assess the equilibrium DNA binding affinity of gapped DNA substrates with pol β (16Prasad R. Beard W.A. Wilson S.H. J. Biol. Chem. 1994; 269: 18096-18101Google Scholar). The assay follows DNA synthesis on a homopolymeric template/primer system and determines the competitive inhibition constant, Ki, for a heteropolymeric DNA substrate that does not support DNA synthesis (i.e. the templating bases on the two competing DNA substrates are different). Fig. 3 illustrates the results from an assay to determine the binding affinity of single-nucleotide gapped DNA that has a G/C or C/C (template/primer) terminus with a templating G or C, respectively. These DNA substrates have similar binding affinities (Table III). The binding affinity of the matched terminus with a templating G is the same as that determined by following the DNA concentration dependence of the pre-steady-state burst amplitude (Kd ∼20 nm) (11Vande Berg B.J. Beard W.A. Wilson S.H. J. Biol. Chem. 2001; 276: 3408-3416Google Scholar). The binding affinities for a series of single-nucleotide gapped DNA substrates with matched or mismatched primer termini were determined and are tabulated in Table III. Although the binding affinities for the one-nucleotide gapped DNA substrates are modestly sensitive to DNA sequence, the mean binding affinities for matched and mismatched primer termini are very similar. Likewise, introduction of an abasic site opposite the primer terminus (i.e. T-1) or an extra base directly behind the primer terminus (+1 frameshift intermediate, P-2) does not significantly affect DNA binding affinity.Table IIIDNA polymerase β equilibrium DNA binding affinity Assays were performed as outlined under "Experimental Procedures." The gapped DNA substrates are illustrated in Fig. 2.Primer terminus(X/Y)n- 1:ZnaIdentity of the template (X)/primer (Y) terminus adjacent to the templating base (Z) at position nKd–dNTP+dNTPnmMatched(G/C):G28 ± 5bMean and S.E. of five independent determinations(G/C):C5(G/C):T8(G/C):GcNucleotide insertion was prevented by using a 3′-deoxycytosine at the primer terminus260.14Mismatched(G/A):C19(G/G):G43(G/G):C50(T/G):C41(T/C):C5(A/A):C140.34(C/C):C163.2Abasic site (T-1)dThe nomenclature for the position of the abasic site adduct is outlined in the legend to Fig. 2C(–/A):C25Frameshift intermediate (P-2)eThis substrate is illustrated in Fig. 2B and does not support DNA synthesis activity (Fig. 5)(G/C):C7a Identity of the template (X)/primer (Y) terminus adjacent to the templating base (Z) at position nb Mean and S.E. of five independent determinationsc Nucleotide insertion was prevented by using a 3′-deoxycytosine at the primer terminusd The nomenclature for the position of the abasic site adduct is outlined in the legend to Fig. 2Ce This substrate is illustrated in Fig. 2B and does not support DNA synthesis activity (Fig. 5) Open table in a new tab To determine crystallographic structures of DNA polymerases with bound substrates, an abortive ternary substrate complex is produced (23Pelletier H. Sawaya M.R. Kumar A. Wilson S.H. Kraut J. Science. 1994; 264: 1891-1903Google Scholar). This is typically achieved with a polymerase-DNA complex, where the first two templating nucleotides are complementary to the included ddNTP. The polymerase inserts the first ddNTP, resulting in a terminated primer and binds another ddNTP, resulting in an abortive ternary complex. Utilizing a 3′-deoxycytosine at the primer terminus, the addition of the complementary dNTP results in a significant increase in the apparent binding affinity for the gapped DNA substrate (Table III), suggesting that important conformational changes have occurred. Pol β-Dependent Mismatch Extension—Since the DNA binding affinities for matched and mismatched primer termini are comparable, the ratio of catalytic efficiencies for correct nucleotide insertion on a mismatched terminus relative to that determined on a matched terminus approximates (fmin0). However, the strong dependence of catalytic efficiency for correct insertion on the identity of the matched primer terminus makes the calculation of (fmin0)potentially ambiguous. For example, the catalytic efficiency for insertion on a G/G mismatch needs to be calculated relative to a matched terminus. In this case, a matched terminus could originate with the template base (i.e. G/C) or, alternatively, the primer base (i.e. C/G). Since the catalytic efficiencies for insertion on these matched termini differ by 25-fold, (fmin0)will differ by 25-fold, depending on the identity of the matched terminus. We have chosen the template base of the mismatch as the reference nucleotide for comparison with the matched terminus, since the template strand serves as the blueprint for DNA synthesis. Consequently, the mismatched primer nucleotide is the incorrect partner. It should be noted, however, that mismatches can arise through correct nucleotide insertion on a misaligned template strand followed by realignment (i.e. dislocation) (24Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Google Scholar). In this scenario, the incorrect nucleotide in the primer strand does not represent a misincorporation event. Steady-state kinetic parameters are tabulated in Table I in order of decreasing catalytic efficiencies for the respective mismatches relative to the corresponding matched terminus. Since insertion of dCTP on a G/G mismatch could not be readily measured (see below), the templating base was changed to thymidine. In general, pol β extends transition intermediates more efficiently than transversions. In most instances, this is due to a decrease in kcat and an increase in Km. However, there are situations where changes in one kinetic parameter dominates over the other. For example, kcat for extension of T/G and T/C is hardly affected relative to T/A, but Km is increased 35- and 74-fold, respectively. The interpretation of these steady-state kinetic parameters is not straightforward due to the observation that product dissociation (i.e. koff for nicked DNA) and nucleotide insertion (i.e. kpol) are partially rate-limiting for correct insertion on a matched terminus (11Vande Berg B.J. Beard W.A. Wilson S.H. J. Biol. Chem. 2001; 276: 3408-3416Google Scholar). Thus, kcat can be a reflection of both rate constants. 3The full expressions for kcat and Km are (kpol koff)/(kpol + koff) and Kd(koff/(kpol + koff)), respectively (25Beard W.A. Wilson S.H. Karn J. HIV: A Practical Approach. 2. IRL Press, New York1995: 15-36Google Scholar). The observation that it is not significantly altered suggests that the increase in Km represents a diminished dNTP binding affinity rather than a change in processivity (kpol/koff). It is not surprising that a mismatched primer terminus results in diminished dNTP binding and insertion, since the primer terminus forms part of the dNTP-binding pocket. However, the kinetic analysis of extension of an A/A or G/G mismatch indicates that the loss of catalytic efficiency is entirely due to the loss of insertion (kpol) without altering the binding affinity for the incoming nucleotide. Because kcat for correct insertion is diminished 460- and 2800-fold respectively, kcat is a direct measure of kpol. Since the DNA binding affinity is not significantly altered (Table III), kpol is now completely rate-limiting. In such a situation, Km is equivalent to Kd for the incoming nucleotide (25Beard W.A. Wilson S.H. Karn J. HIV: A Practical Approach. 2. IRL Press, New York1995: 15-36Google Scholar). The Kd for the correct incoming nucleotide on a matched terminus is ∼10 μm (11Vande Berg B.J. Beard W.A. Wilson S.H. J. Biol. Chem. 2001; 276: 3408-3416Google Scholar, 13Beard W.A. Shock D.D. Yang X.-P. DeLauder S.F. Wilson S.H. J. Biol. Chem. 2002; 277: 8235-8242Google Scholar). Thus, the increase in Km for these mismatche