3 ' + 5 ' Exonucleases of DNA Polymerases E and 6 Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces

The base analog 6-Nhydroxylaminopurine (HAP) induces bidirectional GC + AT and AT + GC transitions that are enhanced in DNA polymerase E and 6 3' + 5' exonuclease-deficient yeast mutants, po12-4 and pol?-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases 5 and E 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+, po12-4 or pol?-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either po12-4 or pol?"Ol, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the urn? alleles placed into chromosome ZZInear to the defined active replication origin ARS?OCi in two orientations indicated that DNA polymerases t and 6 correct HAP-induced DNA replication errors on opposite DNA strands. A variety of prokaryotic and eukaryotic DNA polymerases possess an associated 3' + 5' exonuclease that removes incorrectly inserted nucleotides during DNA synthesis, providing an important step in maintaining high fidelity of genome replication (ECHOLS and GOODMAN 1991; WANG 1991). In the yeast Saccharomyces cerevisiae, three DNA polymerases are essential for chromosomal DNA replication, DNA polymerases a, b and E (SUGINO 1995). The precise role of ach polymerase at a replication fork is unknown. Current models suggest that DNA polymerase a-primase complex initiates DNA synthesis at replication origins and on the lagging strand while DNA polymerases S and E function during elongation. According to one model (MORRISON et al. 1990), DNA polymerase E synthesizes the leading strand of DNA and DNA polymerase S completes the synthesis of Okazaki fragments on the lagging strand. Another model suggests that DNA polymerase E synthesizes Okazaki fragments and DNA polymerase S is responsible for the leading strand synthesis (BURGERS et al. 1991). However, in vitro replication of DNA from the SV40 origin requires only DNA polymerases a and S suggesting that DNA polymerase E might play an essential role in replication-linked DNA repair (WAGA