3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Abstract

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.

Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase

“…Thus, both Orc1p and igin DNA was strikingly similar to that mediated by Pol⑀-HA (Figure 6). At the start of replication, all three proteins one strand of the DNA (Sugino, 1995;Shcherbakova and Pavlov, 1996;Zlotkin et al, 1996). To monitor the began to dissociate from origin DNA and became associated first with nonorigin DNA 8 kb distant from either progress of a replication fork, we analyzed the temporal association of DNA Pol⑀ with DNA sequences spanning origin (305 ϩ 8 kb and 306 ϩ 8 kb) and subsequently with the more distal sequence (17 kb; Figure 6).…”

POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status

“…Model organisms have provided important insights into the role that Pol ε infidelity plays in mutagenesis and tumorigenesis. Studies in yeast have shown that mutation rates are elevated in haploid and diploid strains carrying mutant Pol ε alleles that cause inactivated exonuclease activity (Morrison et al, 1991(Morrison et al, , 1993Morrison and Sugino, 1994;Ohya et al, 2002;Shcherbakova and Pavlov, 1996;Tran et al, 1999). As mutation rates increase, cells can adapt by evolving ways of suppressing this mutagenesis (Dennis et al, 2017;Herr et al, 2011b;Williams et al, 2013).…”

DNA Replication Fidelity: Proofreading in Trans

“…Polymerases d and 3, together with polymerase a (primase), are essential replicative enzymes functioning at DNA replication forks [5]. Point mutations that selectively inactivate the exonuclease domains of polymerases d or 3 confer mutator phenotypes in yeast [6] and mammalian cells [7], and there is good evidence that these exonucleases correct replication errors on opposite DNA strands [8]. It is not clear, however, whether polymerases d and 3 correct only their own errors.…”