Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1.
Fission yeast Cut5/Rad4 plays a unique role in the genome maintenance as it is required for replication, replication checkpoint, and normal UV sensitivity. It is unknown, however, how Cut5 protein is linked to other checkpoint proteins, and what part it plays in replication and UV sensitivity. Here we report that Cut5 interacts with a novel checkpoint protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and human 53BP1 and BRCA1. Crb2 is required for checkpoint arrests induced by irradiation and polymerase mutations, but not for those induced by inhibited nucleotide supply. Upon UV damage, Crb2 is transiently modified, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 modification requires other damage-sensing checkpoint proteins but not Chk1, suggesting that Crb2 acts at the upstream of Chk1. The modified Crb2 exists as a slowly sedimenting form, whereas Crb2 in undamaged cells is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest the cell cycle.
Saka
Y
Y
Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606, Japan.
Esashi
F
F
Matsusaka
T
T
Mochida
S
S
Yanagida
M
M
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000235:genetics; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D017124:Conserved Sequence; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D011503:Transglutaminases; D014466:Ultraviolet Rays
D019313:BRCA1 Protein; D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; C082463:RAD4 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011503:Transglutaminases; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D004259:DNA-Directed DNA Polymerase; D006918:Hydroxyurea