Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

Abstract

We isolated active mutants in Saccharomyces cerevisiae DNA polymerase ␣ that were associated with a defect in error discrimination. Among them, L868F DNA polymerase ␣ has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase ␣. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase ␣-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase ␣ catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3 T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase , and the F34L mutant of S. cerevisiae DNA polymerase has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase ␣ is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.In all organisms with a DNA genome, the genome is replicated by DNA polymerases prior to cell division and one copy is transmitted to each daughter cell. In eukaryotic cells, the DNA polymerase ␣ (pol ␣)-DNA primase complex initiates DNA replication; DNA primase synthesizes a short RNA chain, and pol ␣ extends the RNA primer by ϳ30 deoxyribonucleotides. pol ε and pol ␦ continue DNA synthesis in a processive manner and complete the elongation stage of DNA replication (60).To ensure the survival of each daughter cell, DNA replication must be efficient and accurate. The insertion of the correct nucleotide depends in part on Watson-Crick base pair formation, but this mechanism is not sufficient to obtain high-fidelity DNA replication (23, 30). The observed high fidelity of chromosomal replication is achieved through mechanisms that enhance correct nucleotide insertion and that correct errors after they occur. Eukaryotic pol ␦ and pol ε remove misincorporated nucleotides with an intrinsic 3Ј-5Ј exonuclease activity (37, 38). Some evidence supports the idea of a physical and functional interaction between mismatch repair (MMR) proteins and PCNA, which is an accessory protein of pol ␦ and pol ε (1, 6, 18, 59). Furthermore, polymerase-associated exonucleases may participate in the same pathway that involves MMR (38,39,56). Disruption of this proofreading-MMR pathway causes types of cancers in both humans and mice (13,14,17).Little is known about error prevention and correction mechanisms that enhance the fidelity of DNA pol ␣. pol ␣ lacks 3Ј-5Ј exonuclease activity (36, 46), but it discriminates between correct and incorrect nucleotides during nucleotide incorporation and extension. Mutant polymerase characterization has provided some insight into the mechanism by which nucleotide insertion fidelity is achieved (7,26,41). However, it would be desirable to have a relevant e...

Evidence for Extrinsic Exonucleolytic Proofreading

“…2) between L868M pol α and 5DV pol δ. The fact that mismatch repair remains active in the pol1-L868M-pol3-5DV strain 17,18 argues against this possibility. So too does the fact that no synergy was observed between L868M pol α and exonuclease-deficient pol ε (ref.…”

“…Arguing against this possibility is the fact that mutagenesis in a pol3-5DV strain does not depend on pol ζ, which is renowned for promiscuous mismatch extension. 21,22 Another possibility is suggested by the fact that errors made by L868M pol α are subject to mismatch repair 17,18 and the exonuclease activity of pol δ is implicated in the excision step of mismatch repair. 23 Thus partially defective mismatch repair due to loss of pol δ exonuclease activity could contribute to some of the synergy (Fig.…”

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

“…Because single amino acid changes in Polα, Polδ, and Polε can dramatically affect DNA polymerase fidelity (2,5,6,(22)(23)(24), understanding the functional consequences of naturally occurring variants of these polymerases provides invaluable knowledge of potential cancer risk for individuals with these variants. To this end, we examined reported SNPs and cancer-associated mutations affecting Polδ and Polε.…”

“…Fidelity is further increased in trans through the action of the DNA mismatch repair (MMR) pathway. Mutations affecting the fidelity of yeast Polα, Polδ, and Polε result in a spontaneous mutator phenotype (2)(3)(4)(5)(6). In the case of Polδ, when specific nucleotide selectivity and proofreading defects were engineered in mice, the resulting increased mutation rate was also accompanied by an accelerated tumorigenesis (7)(8)(9).…”

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

“…Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7)(8)(9)(10)(11)(12)(13)(14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporadic hypermutated CRC and EC (20)(21)(22)(23).…”