Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions

Eukaryotic DNA polymerase delta (Pol delta) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion of individual dNTPs opposite a template guanine, Pol delta is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3'-exonuclease activity, implies that Pol delta should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol delta does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations/100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol delta confirm the high nucleotide selectivity of the polymerase and further indicate that proofreading enhances the base substitution fidelity of the wild type enzyme by at least 60-fold. However, Pol delta inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions/100,000 nucleotides polymerized. Moreover, wild type Pol delta frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol delta and Pol epsilon both have high base substitution fidelity, Pol delta is much less accurate than Pol epsilon for deletions involving repetitive sequences. Thus, strand slippage during replication by wild type Pol delta may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these ‘antimutagenic’ changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient ‘mutator’ derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.     

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.     

The high fidelity and unique error signature of human DNA polymerase epsilon

Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3'→5' exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4-5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ.     

The in vitro fidelity of yeast DNA polymerase δ and polymerase ε holoenzymes during dinucleotide microsatellite DNA synthesis

Elucidating the sources of genetic variation within microsatellite alleles has important implications for understanding the etiology of human diseases. Mismatch repair is a well described pathway for the suppression of microsatellite instability. However, the cellular polymerases responsible for generating microsatellite errors have not been fully described. We address this gap in knowledge by measuring the fidelity of recombinant yeast polymerase δ (Pol δ) and ? (Pol ?) holoenzymes during synthesis of a [GT/CA] microsatellite. The in vitro HSV-tk forward assay was used to measure DNA polymerase errors generated during gap-filling of complementary GT(10) and CA(10)-containing substrates and ～90 nucleotides of HSV-tk coding sequence surrounding the microsatellites. The observed mutant frequencies within the microsatellites were 4 to 30-fold higher than the observed mutant frequencies within the coding sequence. More specifically, the rate of Pol δ and Pol ? misalignment-based insertion/deletion errors within the microsatellites was ～1000-fold higher than the rate of insertion/deletion errors within the HSV-tk gene. Although the most common microsatellite error was the deletion of a single repeat unit, ～ 20% of errors were deletions of two or more units for both polymerases. The differences in fidelity for wild type enzymes and their exonuclease-deficient derivatives were ～2-fold for unit-based microsatellite insertion/deletion errors. Interestingly, the exonucleases preferentially removed potentially stabilizing interruption errors within the microsatellites. Since Pol δ and Pol ? perform not only the bulk of DNA replication in eukaryotic cells but also are implicated in performing DNA synthesis associated with repair and recombination, these results indicate that microsatellite errors may be introduced into the genome during multiple DNA metabolic pathways.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are 相似文献   

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.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

The fidelity of DNA synthesis by the catalytic subunit of yeast DNA polymerase alpha alone and with accessory proteins

The fidelity of DNA synthesis catalyzed by the 180-kDa catalytic subunit (p180) of DNA polymerase alpha from Saccharomyces cerevisiae has been determined. Despite the presence of a 3'----5' exonuclease activity (Brooke et al., 1991, J. Biol. Chem., 266, 3005-3015), its accuracy is similar to several exonuclease-deficient DNA polymerases and much lower than other DNA polymerases that have associated exonucleolytic proofreading activity. Average error rates are 1/9900 and 1/12,000, respectively, for single base-substitution and minus-one nucleotide frameshift errors; the polymerase generates deletions as well. Similar error rates are observed with reactions containing the 180-kDa subunit plus an 86-kDa subunit (p86), or with these two polypeptides plus two additional subunits (p58 and p49) comprising the DNA primase activity required for DNA replication. Finally, addition of yeast replication factor-A (RF-A), a protein preparation that stimulates DNA synthesis and has single-stranded DNA-binding activity, yields a polymerization reaction with 7 polypeptides required for replication, yet fidelity remains low relative to error rates for semiconservative replication. The data suggest that neither exonucleolytic proofreading activity, the beta subunit, the DNA primase subunits nor RF-A contributes substantially to base substitution or frameshift error discrimination by the DNA polymerase alpha catalytic subunit.     

In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta

Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable. Strains with same substitution in Rev3, the catalytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Pol alpha (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, -delta, or -epsilon are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Pol alpha mutant has no obvious phenotype. The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Pol delta mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Pol alpha, -delta, and -epsilon mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.     

3'' -> 5'' Exonucleases of DNA Polymerases ε and δ Correct Base Analog Induced DNA Replication Errors on opposite DNA Strands in Saccharomyces Cerevisiae

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC -> AT and AT -> GC transitions that are enhanced in DNA polymerase ε and δ 3' -> 5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ε contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC -> AT and AT -> GC transitions in a Pol(+), pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ε and δ correct HAP-induced DNA replication errors on opposite DNA strands.     

The frameshift infidelity of human DNA polymerase lambda. Implications for function

DNA polymerase lambda (Pol lambda) is a member of the Pol X family having properties in common with several other mammalian DNA polymerases. To obtain clues to possible functions in vivo, we have determined the fidelity of DNA synthesis by human Pol lambda. The results indicate that the average single-base deletion error rate of Pol lambda is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol lambda generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol lambda is different from that of other DNA polymerases and reveals that Pol lambda readily uses template-primers with limited base pair homology at the primer terminus. This ability, together with an ability to fill short gaps in DNA at low dNTP concentrations, is consistent with a role for mammalian Pol lambda in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol lambda has intrinsic 5',2'-deoxyribose-5-phosphate lyase activity.     

Efficiency of correct nucleotide insertion governs DNA polymerase fidelity

DNA polymerase fidelity or specificity expresses the ability of a polymerase to select a correct nucleoside triphosphate (dNTP) from a pool of structurally similar molecules. Fidelity is quantified from the ratio of specificity constants (catalytic efficiencies) for alternate substrates (i.e. correct and incorrect dNTPs). An analysis of the efficiency of dNTP (correct and incorrect) insertion for a low fidelity mutant of DNA polymerase beta (R283A) and exonuclease-deficient DNA polymerases from five families derived from a variety of biological sources reveals that a strong correlation exists between the ability to synthesize DNA and the probability that the polymerase will make a mistake (i.e. base substitution error). Unexpectedly, this analysis indicates that the difference between low and high fidelity DNA polymerases is related to the efficiency of correct, but not incorrect, nucleotide insertion. In contrast to the loss of fidelity observed with the catalytically inefficient R283A mutant, the fidelity of another inefficient mutant of DNA polymerase beta (G274P) is not altered. Thus, although all natural low fidelity DNA polymerases are inefficient, not every inefficient DNA polymerase has low fidelity. Low fidelity polymerases appear to be an evolutionary solution to how to replicate damaged DNA or DNA repair intermediates without burdening the genome with excessive polymerase-initiated errors.     

Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).