The S/M checkpoint at 37 degrees C and the recovery of viability of the mutant pol delta ts3 require the crb2(+)/rhp9(+) gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled f rom completion of DNA replication when DNA synthesis is impaired by a therm osensitive mutation in the gene encoding the catalytic subunit of DNA polym erase delta. By functional complementation, we cloned the wild-type gene an d identified it as the recently cloned checkpoint gene crb2(+)/rhp9(+). Thi s gene has been implicated in the DNA damage checkpoint and acts in the Chk 1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment a t 30 degrees C, but were defective in this checkpoint function when treated with MMS at 37 degrees C. We analysed the involvement of Crb2 in the S/M c heckpoint by blocking DNA replication with hydroxyurea, by using S phase cd c mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37 degrees C. Furthermore, th e crb2(+) gene was required, together with the cds1(+) gene, for the S/M ch eckpoint at 30 degrees C. Finally, both the crb2 deletion and the crb2-1 al lele induced a rapid death phenotype in the pol delta ts3 background at bot h 30 degrees C and 37 degrees C. The rapid death phenotype was independent of the checkpoint functions.