DAMAGE AND REPLICATION CHECKPOINT CONTROL IN FISSION YEAST IS ENSUREDBY 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 U V sensitivity. Here we report that Cut5 interacts with a novel checkpo int protein Crb2 and that this interaction is needed for normal genome maintenance. The carboxyl terminus of Crb2 resembles yeast Rad9 and h uman 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 modif ied, probably phosphorylated, with a similar timing of phosphorylation in Chk1 kinase, which is reported to restrain Cdc2 activation. Crb2 m odification 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 ce lls is in a rapidly sedimenting structure. Cut5 and Crb2 interact with Chk1 in a two-hybrid system. Moreover, moderate overexpression of Chk 1 suppresses the phenotypes of cut5 and crb2 mutants. Cut5, Crb2, and Chk1 thus may form a checkpoint sensor-transmitter pathway to arrest t he cell cycle.