DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s−1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive

Kinetics of error generation in homologous B-family DNA polymerases

The kinetics of forming a proper Watson–Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all ∼2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase α, δ, ɛ or ζ, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure–function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 Å crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol

An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol zeta (a family-B translesion synthesis polymerase). Inactivation of the 3'–5' proofreading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with amarked tendency tomake changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an errorprone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA

DNA damage alters DNA polymerase δ to a form that exhibits increased discrimination against modified template bases and mismatched primers

Human DNA polymerase δ (Pol δ4), a key enzyme in chromosomal replication, is a heterotetramer composed of the p125, p50, p68 and p12 subunits. Genotoxic agents such as UV and alkylating chemicals trigger a DNA damage response in which Pol δ4 is converted to a trimer (Pol δ3) by degradation of p12. We show that Pol δ3 has altered enzymatic properties: it is less able to perform translesion synthesis on templates containing base lesions (O6-MeG, 8-oxoG, an abasic site or a thymine-thymine dimer); a greater proofreading activity; an increased exonuclease/polymerase activity ratio; a decreased tendency for the insertion of wrong nucleotides, and for the extension of mismatched primers. Overall, our findings indicate that Pol δ3 exhibits an enhanced ability for the detection of errors in both primers and templates over its parent enzyme. These alterations in Pol δ3 show that p12 plays a major role in Pol δ4 catalytic functions, and provides significant insights into the rationale for the conversion of Pol δ4 to Pol δ3 in the cellular response to DNA damage

Mutator Suppression and Escape from Replication Error–Induced Extinction in Yeast

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10−3 inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase ζ

To probe Pol ζ functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol ζ in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol ζ is 30% as active as wild-type Pol ζ when replicating undamaged DNA. L979F Pol ζ shares with wild-type Pol ζ the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol ζ is error-prone compared to wild-type Pol ζ, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol ζ in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol ζ also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol ζ, and perhaps wild-type Pol ζ, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo

DNA polymerase ɛ and δ exonuclease domain mutations in endometrial cancer

Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ɛ, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5–10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity