The Schizosaccharomyces pombe cdt2(+) gene, a target of G1-S phase-specific transcription factor complex DSC1, is required for mitotic and premeiotic DNA replication.
We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1(+)/cdt2(+) was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2(+) was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2(+) expression in vivo, and a cdt2(+) promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30 degrees and were unable to grow at 19 degrees. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22(+), a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19 degrees. These observations suggest that cdt2(+) plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.
Yoshida
Shu-hei
SH
Department of Biology, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan.
Al-Amodi
Hiba
H
Nakamura
Taro
T
McInerny
Christopher J
CJ
Shimoda
Chikashi
C
D015112:4-Nitroquinoline-1-oxide; D000595:Amino Acid Sequence; D002166:Camptothecin; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D016521:Electrophoresis, Gel, Pulsed-Field; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D015870:Gene Expression; D006918:Hydroxyurea; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D015112:4-Nitroquinoline-1-oxide; D006918:Hydroxyurea; D002166:Camptothecin