Characterization of Schizosaccharomyces pombe Pfh1 DNA helicase activity and its genetic interaction with Dna2, Cdc24 and pol delta

초록

The Schizosaccharomyces pombe pfh1+ gene encoding a DNA helicase suppresses a temperature-sensitive (ts) mutant allele of cdc24+ that functions with dna2+ in Okazaki fragment processing. In this report, we examined enzymatic properties of S. pombe Pfh1 helicase and genetic interactions among pfh1+, cdc24+, and dna2+. Pfh1 possesses intrinsic helicase and DNA-dependent ATPase activities and is active as monomer. The helicase activity of Pfh1 displaced only short (<30-base pair) duplex DNA efficiently in a highly distributed manner and was markedly stimulated by a replication-fork structure in the substrate. The ts phenotype of dna2-C2 mutant or cdc24-M38 was suppressed when it was combined with pfh1-R20. Overexpression of wild type pfh1+ in cdc24-M38 pfh1-R20 or dna2-C2 pfh1-R20 double mutant cells prevented either pfh1-R20 or pfh1-R23 from suppressing the ts growth defect of cdc24-M38 or dna2-C2. This indicates that simultaneous loss-of-function mutations of pfh1+ and dna2+ (or cdc24+) are essential to restore growth defect. In support of this, Pfh1-R20 mutant protein purified displayed significantly reduced ATPase and helicase activities. We detected a synthetic lethal or growth defect between pfh1-R20 and cdc27 or pol 3 genes encoding pol delta. Based upon our genetic and biochemical data, the role played by Pfh1 in conjunction with Dna2-Cdc24 and pol delta during Okazaki fragment processing is discussed.