Nucleic Acids Research, 2002, Vol. 30, No. 21 4728-4739
© 2002 Oxford University Press
The fission yeast pfh1+ gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase
1 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK, 2 Department of Molecular Oncology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8519, Japan, 3 National Creative Research Initiative Center for Cell Cycle Control, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchun-Dong, Changan-Ku, Suwon-Si, Kyunggi-Do, 440-746, South Korea and 4 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-Ku, Tokyo 113-0033, Japan
*To whom correspondence should be addressed at Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK. Tel: +44 131 650 7085; Fax: +44 131 650 8650; Email: hiroyuki.tanaka{at}ed.ac.uk
Present address:
Koichi Tanaka, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030, Vienna, Austria
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 comple tion 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.
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