Replicative DNA polymerase ? but not ? proofreads errors in Cis and in Trans.

ABSTRACT: It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ? and of the lagging strand by DNA polymerase ?. However, the role of Pol ? in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ? or Pol ? in diploid strains. We find that wild-type Pol ? molecules cannot proofread errors created by proofreading-defective Pol ? molecules, whereas Pol ? can not only proofread errors created by proofreading-defective Pol ? molecules, but can also proofread errors created by Pol ?-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol ? and also explain the higher mutation rates observed in Pol ?-proofreading mutants compared to Pol ?-proofreading defective mutants. For strains reverting via AT?GC, TA?GC, CG?AT, and GC?AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3'-terminal 8 oxoG, unlike a 3'-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.

SUBMITTER: Flood CL 

PROVIDER: S-EPMC4351087 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature