DNA polymerase leading strand signature mutations result from defects in its proofreading activity

The evidence that purified pol2-M644G DNA polymerase (Pol)epsilon exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Pol epsilon mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Pol epsilon in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Pol epsilon proofreading activity by analyzing their rate in Pol epsilon proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Pol epsilon exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Pol epsilon replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Pol zeta in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Pol epsilon proofreading activity and not from the role of Pol epsilon as a leading strand replicase, and it conforms with the genetic evidence for a major role of Pol delta in replication of both the DNA strands.