Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast.
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, cdc27 or pol 3, 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 -mediated lagging strand displacement DNA synthesis.
National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Korea.
Gi-Hyuck
GH
Ryu
Hiroyuki
H
Tanaka
Do-Hyung
DH
Kim
Jeong-Hoon
JH
Kim
Sung-Ho
SH
Bae
Young-Nam
YN
Kwon
Joon Shick
JS
Rhee
Stuart A
SA
MacNeill
Yeon-Soo
YS
Seo
D000251:Adenosine Triphosphatases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins; D005800:Genes, Fungal; D009154:Mutation; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000235:genetics; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity; D013489:Suppression, Genetic; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics; D035602:Transcriptional Elongation Factors
C468288:Cdc24 protein, S pombe; D018797:Cell Cycle Proteins; D029968:Escherichia coli Proteins; C022262:Okazaki fragments; D010445:Peptide Elongation Factors; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C468287:Pfh1 protein, S pombe