Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

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The Schizosaccharomyces pombe pfh1+ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1(+) gene suppress a temperature-sensitive allele of cdc24(+), which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27or pol3,all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1(+) abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1(+) and dna2' (or cdc24) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol &delta;-mediated lagging strand displacement DNA synthesis.
Publisher
Oxford Univ Press
Issue Date
2004-08
Language
English
Article Type
Article
Keywords

SACCHAROMYCES-CEREVISIAE DNA2; SCHIZOSACCHAROMYCES-POMBE; REPLICATION FORK; ESSENTIAL 5&apos; -DNA; 3&apos; -DNA HELICASE; SIMIAN VIRUS-40; LAGGING-STRAND; S-PHASE; COMPLETION; ENCODES

Citation

NUCLEIC ACIDS RESEARCH, v.32, no.14, pp.4205 - 4216

ISSN
0305-1048
DOI
10.1093/nar/gkh720
URI
http://hdl.handle.net/10203/83076
Appears in Collection
BS-Journal Papers(저널논문)
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