DNA polymerase beta fidelity: halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal differences at the chemical transition state

The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.     

Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5'',5''''''-adenosyl) tetraphosphate.

Six new methylenephosphonate analogues of P1P4-bis-(5',5'-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.     

Recognition of beta beta'-substituted and alpha beta,alpha'beta'-disubstituted phosphonate analogues of bis(5'-adenosyl) tetraphosphate by the bis(5'-nucleosidyl)-tetraphosphate pyrophosphohydrolases from Artemia embryos and Escherichia coli

A total of 13 phosphonate analogues of bis(5'-adenosyl) tetraphosphate (AppppA) have been tested as substrates and inhibitors of the asymmetrically cleaving bis(5'-nucleosidyl) tetraphosphatase (NppppNase) from Artemia and the symmetrically cleaving NppppNase from Escherichia coli. With the Artemia enzyme, the substrate efficiency of beta beta'-substituted compounds decreased with decreasing substituent electronegativity (O greater than CF2 greater than CHF greater than CCl2 greater than CHCl greater than CH2) such that AppCF2ppA and AppCH2ppA were hydrolyzed at 70% and 2.5% of the rate of AppppA, respectively. These compounds were competitive inhibitors of this enzyme with Ki values that generally also decreased with electronegativity from 12 microM for AppCF2ppA to 0.4 microM for AppCH2ppA (Km for AppppA = 33 microM). AppCH = CHppA and AppCH2CH2ppA were neither effective substrates nor inhibitors of the Artemia enzyme. Alpha beta,alpha'beta'-Disubstituted analogues were generally less effective inhibitors with Ki values ranging from 23 microM (ApCH2ppCH2pA) to greater than 1.5 mM (ApCH2CH2ppCH2CH2pA). However, they displayed a low and unexpected rate of symmetrical cleavage by the Artemia enzyme: e.g., ApCHFppCHFpA yielded ApCHFp at 3% of the rate of AppppA breakdown. Both sets of analogues were also competitive inhibitors of the E. coli NppppNase with Ki values ranging from 7 microM (AppCH2ppA) to 250 microM (ApCH2CH2ppCH2CH2pA) (Km for AppppA = 28 microM). The only alpha beta,alpha'beta'-disubstituted analogue to be hydrolyzed by the E. coli enzyme was ApCF2ppCF2pA at 0.2% of the rate of AppppA; however, several of the beta beta'-substituted compounds showed a limited degree of asymmetrical cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition

The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase alpha, and HeLa polymerase beta to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2',3'-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP greater than ACVTP approximately equal to DHPGTP greater than ddGTP; polymerase alpha, dGTP greater than ACVTP approximately equal to DHPGTP much greater than ddGTP; polymerase beta, ddGTP greater than dGTP much greater than ACVTP approximately equal to DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase alpha. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase alpha, although there was a considerable reduction in the efficiency of utilization of the 3'-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase alpha, a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.     

Interaction of soluble pig heart glutamate-aspartate transaminase with various beta,gamma-unsaturated amino acids

beta-Ethylidene-DL-aspartate (beta EA) and beta-methylene-DL-glutamate (beta MG) were synthesized and tested as potential suicide inhibitors of soluble pig heart glutamate-aspartate transaminase (sGAT). beta MG was found to be a) a substrate with a very low turnover number relative to glutamate and b) a competitive inhibitor with respect to aspartate (albeit with a large binding constant). At high concentrations beta MG inactivated the enzyme but only very slowly. beta EA was also found to be a substrate with a very low turnover number; it did not inactivate the enzyme (1 hr, 25 degrees C) even at a high concentration. However, beta EA was found to bind to the enzyme with an affinity comparable to that of aspartate and glutamate. beta-Methylene-DL-aspartate (beta MA) has been shown to rapidly inactivate glutamate-aspartate transaminase. Therefore, it appears that glutamate-aspartate transaminase can bind analogues of aspartate with alkene groups in the beta position. The conjugated carbonyl groups of beta MA and beta EA will enhance Michael addition in comparison with that expected for vinylglycine. On the other hand, the presence of the methyl groups should reduce the electrophilicity of the double bond of beta EA compared to beta MA. This deactivation and/or steric hindrance to Michael attack may account for the inability of beta EA to inactivate sGAT. Therefore, it may be possible to design selective suicide inhibitors of glutamate-aspartate++ transaminase with the following structure: HO2CC(= CHX)CH(CO2H)NH2, where X is an electron-withdrawing group. Ideally, X would increase the reactivity of the double bond while affording a minimum of steric hindrance to susceptible enzyme-bound bases.     

Exploring the role of large conformational changes in the fidelity of DNA polymerase beta

The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase beta are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.     

Substrate and inhibitory specificity of riboflavin kinase from Pichia guilliermondii yeast]

The interaction of purified riboflavin kinase (EC 2.7.1.26) from Pichia guilliermondii with 44 structural vitamin B2 analogues is studied. The presence of D-ribityl lateral chain in an analogue structure is found to be necessary for the substrate activity. The substitution of CH3 groups in the 7 and 8 positions of isoalloxazine ring in the riboflavin molecule for CF3, Cl, H, NH2 and N(CH3)2 resulted in the decrease of the analogue affinity to riboflavin kinase as compared with the natural substrate, vitamin B2. The most efficient enzyme inhibitors of analogues without substrate properties turned to be trifluoromethylisoalloxazines, containing 2'-hydroxyethyl group at N10. The elongation of D-ribityl lateral chain, the elimination of change of CH3-groups in the 7 and 8 positions for CF3- Cl-, COOH-substitutors resulted in the decrease of the inhibitory effect of flavines. Modifications in the structure of isoalloxazine ring, etherification of OH-groups in the lateral D-ribityl chain, and the introduction of volume substitutors (N-piperidyl, D-ribitylamine, hydroxyethylamine) prevented the interaction of the analogue with riboflavin kinase. Flavin nucleotides (FMN and FAD) did not affect the rate of vitamin B2 phosphorylation.     

Nucleoside 5'-(beta, gamma-peroxytriphosphates)

Procedures for the synthesis, purification, and characterization of beta, gamma-peroxy analogues of the eight common ribo- and deoxyribonucleoside triphosphates have been developed. Although adenosine 5'-(beta, gamma-peroxytriphosphate) was stable to conditions in most biochemical systems, incubation of a solution of the analogue at 100 degrees C led to formation of AMP and ATP, as well as ADP. NAD+ pyrophosphorylase was the only enzyme among 13 tested for which adenosine 5'-(beta, gamma-peroxytriphosphate) was a good substrate, but the analogue was an effective inhibitor for a number of kinases. The peroxy compounds tested inactive with Escherichia coli RNA polymerase and DNA polymerase I, as well as with wheat germ RNA polymerase II.     

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.     

Fidelity of the human mitochondrial DNA polymerase

We have quantified the fidelity of polymerization of DNA by human mitochondrial DNA polymerase using synthetic DNA oligonucleotides and recombinant holoenzyme and examining each of the possible 16-base pair combinations. Although the kinetics of incorporation for all correct nucleotides are similar, with an average Kd of 0.8 microM and an average k(pol) of 37 s(-1), the kinetics of misincorporation vary widely. The ground state binding Kd of incorrect bases ranges from a low of 25 microM for a dATP:A mispair to a high of 360 microM for a dCTP:T mispair. Similarly, the rates of incorporation of incorrect bases vary from 0.0031 s(-1) for a dCTP:C mispair to 1.16 s(-1) for a dGTP:T mispair. Due to the variability in the kinetic parameters for misincorporation, the estimates of fidelity range from 1 error in 3563 nucleotides for dGTP:T to 1 error in 2.3 x 10(6) nucleotides for dCTP:C. Interestingly, the discrimination against a dGTP:T mismatch is 16.5 times lower than that of a dTTP:G mismatch due to a tighter Kd for ground state binding and a faster rate of incorporation of the dGTP:T mismatch relative to the dTTP:G mismatch. We calculate an average fidelity of 1 error in 440,000 nucleotides.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.     

Analysis of inhibitors of bacteriophage T4 DNA polymerase.

Bacteriophage T4 DNA polymerase was inhibited by butylphenyl nucleotides, aphidicolin and pyrophosphate analogs, but with lower sensitivities than other members of the B family DNA polymerases. The nucleotides N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butylanilino)dATP (BuAdATP) inhibited T4 DNA polymerase with competitive Ki values of 0.82 and 0.54 microM with respect to dGTP and dATP, respectively. The same compounds were more potent inhibitors in truncated assays lacking the competitor dNTP, displaying apparent Ki values of 0.001 and 0.0016 microM, respectively. BuPdGTP was a substrate for T4 DNA polymerase, and the resulting 3'-BuPdG-primer:template was bound strongly by the enzyme. Each of the non-substrate derivatives, BuPdGDP and BuPdGMPCH2PP, inhibited T4 DNA polymerase with similar potencies in both the truncated and variable competitor assays. These results indicate that BuPdGTP inhibits T4 DNA polymerase by distinct mechanisms depending upon the assay conditions. Reversible competitive inhibition predominates in the presence of dGTP, and incorporation in the absence of dGTP leads to potent inhibition by the modified primer:template. The implications of these findings for the use of these inhibitors in the study of B family DNA polymerases is discussed.     

Loop II of DNA polymerase beta is important for discrimination during substrate binding

Loop II of DNA polymerase beta (pol β) consists of 14 amino acid residues and is highly flexible and solvent exposed. Previous research from our laboratory has shown that this loop is important for polymerase activity and fidelity. In the study presented here, we demonstrate that a shortened five amino acid residue loop compromises the fidelity of pol β. This five-residue loop, termed ENEYP, induces one base frameshift errors and A–C transversions within a specific sequence context. We demonstrate that ENEYP misincorporates dGTP opposite template A at higher efficiencies than wild-type pol β. The kinetic basis for misincorporation is a defect in discrimination of the correct from incorrect dNTP substrate at the level of ground-state binding. Our results are consistent with the idea that loop II of pol β functions to maintain accurate DNA synthesis by a direct or indirect influence on the nucleotide binding pocket.     

Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase.

The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10(-3)) were observed at this (guanine) site. We sought evidence for RNA 3'-->5' exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[1-thio]triphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.     

Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues.

Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme E1 is accompanied by ATP hydrolysis to yield AMP and PPi, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pi. We show here that either of two beta, gamma-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a beta, gamma-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, E1 has apparent Km and Vmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the beta, gamma-imido analogue preferable. In one application, beta, gamma-imido-ATP was used in combination with ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E. (1989) J. Biol. Chem. 264, 16739-16747] to show that conjugate yields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependent proteolysis. In a second application, ATP and beta, gamma-imido-ATP were compared in a pulse-chase experiment to investigate the contributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability.     

Mismatched and matched dNTP incorporation by DNA polymerase beta proceed via analogous kinetic pathways

While matched nucleotide incorporation by DNA polymerase beta (Pol beta) has been well-studied, a true understanding of polymerase fidelity requires comparison of both matched and mismatched dNTP incorporation pathways. Here we examine the mechanism of misincorporation for wild-type (WT) Pol beta and an error-prone I260Q variant using stopped-flow fluorescence assays and steady-state fluorescence spectroscopy. In stopped-flow, a biphasic fluorescence trace is observed for both enzymes during mismatched dNTP incorporation. The fluorescence transitions are in the same direction as that observed for matched dNTP, albeit with lower amplitude. Assignments of the fast and slow fluorescence phases are designated to the same mechanistic steps previously determined for matched dNTP incorporation. For both WT and I260Q mismatched dNTP incorporation, the rate of the fast phase, reflecting subdomain closing, is comparable to that induced by correct dNTP. Pre-steady-state kinetic evaluation reveals that both enzymes display similar correct dNTP insertion profiles, and the lower fidelity intrinsic to the I260Q mutant results from enhanced efficiency of mismatched incorporation. Notably, in comparison to WT, I260Q demonstrates enhanced intensity of fluorescence emission upon mismatched ternary complex formation. Both kinetic and steady-state fluorescence data suggest that relaxed discrimination against incorrect dNTP by I260Q is a consequence of a loss in ability to destabilize the mismatched ternary complex. Overall, our results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol beta originates from destabilization of the mismatched closed ternary complex and chemical transition state.     

Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis

We report the crystal structures of a translesion DNA polymerase, Dpo4, complexed with a matched or mismatched incoming nucleotide and with a pyrophosphate product after misincorporation. These structures suggest two mechanisms by which Dpo4 may reject a wrong incoming nucleotide with its preformed and open active site. First, a mismatched replicating base pair leads to poor base stacking and alignment of the metal ions and as a consequence, inhibits incorporation. By replacing Mg2+ with Mn2+, which has a relaxed coordination requirement and tolerates misalignment, the catalytic efficiency of misincorporation increases dramatically. Mn2+ also enhances translesion synthesis by Dpo4. Subtle conformational changes that lead to the proper metal ion coordination may, therefore, be a key step in catalysis. Second, the slow release of pyrophosphate may increase the fidelity of Dpo4 by stalling mispaired primer extension and promoting pyrophosphorolysis that reverses the polymerization reaction. Indeed, Dpo4 has robust pyrophosphorolysis activity and degrades the primer strand in the presence of pyrophosphate. The correct incoming nucleotide allows DNA synthesis to overcome pyrophosphorolysis, but an incorrect incoming nucleotide does not.     

Kinetic analysis of base substitution mutagenesis by transient misalignment of DNA and by miscoding

We measured the insertion fidelity of DNA polymerases alpha and beta and yeast DNA polymerase I at a template site that was previously observed to yield a high frequency of T----G transversions when copied by DNA polymerase beta but not by the other two polymerases. The results provide direct biochemical evidence that base substitution errors by DNA polymerase beta can result from a dislocation mechanism governed by DNA template-primer misalignment. In contrast to DNA polymerase beta, neither Drosophila DNA polymerase alpha nor yeast DNA polymerase I appear to misinsert nucleotides by a dislocation mechanism in either the genetic or kinetic fidelity assays. Dislocation errors by DNA polymerase beta are characterized primarily by a substantial reduction in the apparent Km for inserting a "correct," but ultimately errant, nucleotide compared to the apparent Km governing direct misinsertion. For synthesis by DNA polymerase beta, dislocation results in a 35-fold increase in dCMP incorporation opposite template T (T----G transversion) and a 20-35-fold increase in dTMP incorporation opposite T (T----A transversion); these results are consistent with parallel genetic fidelity measurements. DNA polymerase beta also produces base substitution errors by direct misinsertion. Here nucleotide insertion fidelity results from substantial differences in both Km and Vmax for correct versus incorrect substrates and is influenced strongly by local base sequence.