Abstract
The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adenosine Triphosphatases / genetics
-
Adenosine Triphosphatases / isolation & purification
-
Adenosine Triphosphatases / metabolism
-
Alleles
-
Catalysis
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / isolation & purification
-
Cell Cycle Proteins / metabolism
-
Cell Nucleus / metabolism
-
Conserved Sequence
-
DNA Damage / drug effects
-
DNA Helicases / genetics
-
DNA Helicases / isolation & purification
-
DNA Helicases / metabolism*
-
DNA Helicases / pharmacology
-
DNA Repair
-
DNA Replication
-
DNA, Fungal / analysis
-
G2 Phase
-
Gene Deletion
-
Genes, Essential / genetics*
-
Genes, Fungal / genetics
-
Hydroxyurea / pharmacology
-
Methyl Methanesulfonate / pharmacology
-
Nuclear Proteins / genetics
-
Nuclear Proteins / metabolism
-
RNA, Fungal / genetics
-
RNA, Fungal / metabolism
-
S Phase* / drug effects
-
Schizosaccharomyces / cytology*
-
Schizosaccharomyces / drug effects
-
Schizosaccharomyces / enzymology*
-
Schizosaccharomyces / genetics
-
Schizosaccharomyces pombe Proteins / genetics
-
Schizosaccharomyces pombe Proteins / isolation & purification
-
Schizosaccharomyces pombe Proteins / metabolism*
-
Schizosaccharomyces pombe Proteins / pharmacology
-
Suppression, Genetic
-
Temperature
Substances
-
Cdc24 protein, S pombe
-
Cell Cycle Proteins
-
DNA, Fungal
-
Nuclear Proteins
-
RNA, Fungal
-
Schizosaccharomyces pombe Proteins
-
Methyl Methanesulfonate
-
Adenosine Triphosphatases
-
DNA Helicases
-
Pfh1 protein, S pombe
-
Hydroxyurea