Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae.
Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta 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 delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.
Shimoda
Chikashi
C
Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan. shimoda@sci.osaka-cu.ac.jp
Itadani
Akiko
A
Sugino
Akio
A
Furusawa
Mitsuru
M
D004258:DNA Polymerase III / Q000235:genetics*; D005808:Genes, Recessive; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013696:Temperature
D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III