The S/M checkpoint at 37°C and the recovery of viability of the mutant polδts3 require the crb2⁺/rhp9⁺ gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase δ. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2⁺/rhp9⁺. This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 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 at 30°C, but were defective in this checkpoint function when treated with MMS at 37°C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37°C. Furthermore, the crb2⁺ gene was required, together with the cds1⁺ gene, for the S/M checkpoint at 30°C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the polδts3 background at both 30°C and 37°C. The rapid death phenotype was independent of the checkpoint functions.