Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase δ as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases 6 and F, both of which are required for chromosomal DNA replication, contain proofreading 3'-> 5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong imitators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase 8 variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA 8 polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.