NMR investigation of primer-template models: structural effect of sequence downstream of a thymine template on mutagenesis in DNA replication

Misaligned structures can occur in primer-templates during DNA replication, which can be bypassed and extended by low-fidelity polymerases and ultimately lead to mutations. In this study, we have investigated how the nucleotide downstream of a thymine template affects the primer-template structures upon misincorporation of dNTPs. The base pair structures at the replicating sites of a set of primer-template models containing either a G or an A downstream of the thymine template have been determined using NMR spectroscopy. Incorporation of dCTP and dTTP opposite 5'-GT and 5'-AT templates, respectively, can result in misaligned structures with a T-bulge. Depending on the downstream sequence, subsequent extension of the primers may stabilize the misaligned structures or cause the formation of mismatched structures. These results provide alternative pathways for base substitution and deletion errors during DNA replication by low-fidelity polymerases.     

Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.     

Human DNA polymerase iota utilizes different nucleotide incorporation mechanisms dependent upon the template base

Human DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Polι is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Polι, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Polι dependent upon the template base and that when T is the templating base, Polι accommodates the wobble base pair better than the Watson-Crick base pair.     

Preferential incorporation of G opposite template T by the low-fidelity human DNA polymerase iota

DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Pol(iota), encoded by the RAD30B gene. We show that human Pol(iota) violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Pol(iota) preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Pol(iota) compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Pol(iota) to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Pol(iota) copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Pol(iota) incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Pol(iota) in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Pol(iota) may additionally play a specialized function in human biology.     

Structure of purine-purine mispairs during misincorporation and extension by Escherichia coli DNA polymerase I

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.     

Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.     

B family DNA polymerases asymmetrically recognize pyrimidines and purines

We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.     

Nucleotide selection by the Y-family DNA polymerase Dpo4 involves template translocation and misalignment

Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.     

Oxidation of thymine to 5-formyluracil in DNA promotes misincorporation of dGMP and subsequent elongation of a mismatched primer terminus by DNA polymerase

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3'-5'-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f(A) = V(max)/K(m)) decreased to (1/4) to (1/2), depending on the nearest neighbor base pair, and that of dGMP (f(G)) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f(G)/f(A) for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK(a) = 8.6) but not T (pK(a) = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.     

The biochemical basis of 5-bromouracil-induced mutagenesis. Heteroduplex base mispairs involving bromouracil in G x C----A x T and A x T----G x C mutational pathways

We have investigated the mechanism of bromouracil-induced transition mutations in vitro using synthetic DNA templates and purified T4 DNA polymerase. Evidence is presented for the occurrence of bromouracil-guanine base pairs in product DNA in the G x C----A x T pathway where guanine is present in the DNA template and bromouracil is present as the deoxynucleoside triphosphate substrate 5-bromodeoxyuridine triphosphate. This finding supports a widely known but as yet untested model proposed by Freese (Freese, E. (1959) J. Mol. Biol. 1, 87-105) in which bromouracil-guanine base pairs are intermediates in 5-bromodeoxyuridine-induced transition mutation pathways. We find that the newly formed B x G base pairs are proofread with an efficiency of 75-85% by the 3' -exonuclease of T4 polymerase. The insertion of bromouracil occurring in direct competition with cytosine deoxyribonucleotides opposite template guanine sites is 1.1 +/- 0.14% (mean +/- S.E.), and the misincorporation ratio, inc(B)/inc(C), is reduced 6-fold by the action of the proofreading exonuclease to 0.16 +/- 0.02% (mean +/- S.E.). A previous study by Trautner et al. (Trautner, T. A., Swartz, M. N., and Kornberg, A. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 449-455) suggested that, while template bromouracil stimulates incorporation of dGMP in the A x T----G x C transition mutation pathway, it may not be occurring exclusively by the pathway proposed by Freese. We concur with these earlier results, and, in addition, we find the surprising result that the 3'-exonuclease activity of wild-type T4 polymerase removes little or no incorporated dGMP on bromouracil-containing templates.     

Mismatch-induced conformational distortions in polymerase beta support an induced-fit mechanism for fidelity

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

Structural Basis for Promutagenicity of 8-Halogenated Guanine

8-Halogenated guanine (haloG), a major DNA adduct formed by reactive halogen species during inflammation, is a promutagenic lesion that promotes misincorporation of G opposite the lesion by various DNA polymerases. Currently, the structural basis for such misincorporation is unknown. To gain insights into the mechanism of misincorporation across haloG by polymerase, we determined seven x-ray structures of human DNA polymerase β (polβ) bound to DNA bearing 8-bromoguanine (BrG). We determined two pre-catalytic ternary complex structures of polβ with an incoming nonhydrolyzable dGTP or dCTP analog paired with templating BrG. We also determined five binary complex structures of polβ in complex with DNA containing BrG·C/T at post-insertion and post-extension sites. In the BrG·dGTP ternary structure, BrG adopts syn conformation and forms Hoogsteen base pairing with the incoming dGTP analog. In the BrG·dCTP ternary structure, BrG adopts anti conformation and forms Watson-Crick base pairing with the incoming dCTP analog. In addition, our polβ binary post-extension structures show Hoogsteen BrG·G base pair and Watson-Crick BrG·C base pair. Taken together, the first structures of haloG-containing DNA bound to a protein indicate that both BrG·G and BrG·C base pairs are accommodated in the active site of polβ. Our structures suggest that Hoogsteen-type base pairing between G and C8-modified G could be accommodated in the active site of a DNA polymerase, promoting G to C mutation.     

Structural basis of error‐prone replication and stalling at a thymine base by human DNA polymerase ι

Human DNA polymerase ι (polι) is a unique member of Y‐family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson–Crick base pairing by polι. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H‐bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template ‘U‐turn’ is stabilized by polι and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain‐swapping experiments indicate that the finger domain is responsible for polι's high error rates on pyrimidines and determines the incorporation specificity.     

An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.     

Structural effect of the anticancer agent 6-thioguanine on duplex DNA

The incorporation of 6-thioguanine (S6G) into DNA is an essential step in the cytotoxic activity of thiopurines. However, the structural effects of this substitution on duplex DNA have not been fully characterized. Here, we present the solution structures of DNA duplexes containing S6G opposite thymine (S6G·T) and opposite cytosine (S6G·C), solved by high-resolution NMR spectroscopy and restrained molecular dynamics. The data indicate that both duplexes adopt right-handed helical conformations with all Watson–Crick hydrogen bonding in place. The S6G·T structures exhibit a wobble-type base pairing at the lesion site, with thymine shifted toward the major groove and S6G displaced toward the minor groove. Aside from the lesion site, the helices, including the flanking base pairs, are not highly perturbed by the presence of the lesion. Surprisingly, thermal dependence experiments suggest greater stability in the S6G-T mismatch than the S6G-C base pair.     

Loss of DNA minor groove interactions by exonuclease-deficient Klenow polymerase inhibits O6-methylguanine and abasic site translesion synthesis

The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.     

The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.