CHARACTERIZATION OF THE CHECKPOINT GENE RAD53 MEC2 IN SACCHAROMYCES-CEREVISIAE/

Saccharomyces cevevisiae cells carrying mutations in RAD53/MEC2 fail t o arrest in the S phase when DNA replication is blocked (the S/M check point) or in the G2 phase when DNA is damaged (the G2/M checkpoint). W e isolated and determined the DNA sequence of RAD53 and found that it is identical to the SPK1 gene previously identified by Stern et al. (1 991). In addition to its checkpoint functions, we show here that RAD53 is essential for cell viability because null mutants are inviable. We ak genomic suppressors of the essential function do arise frequently, though they do not suppress the checkpoint defects of the null mutant. This genetically separates the essential and checkpoint functions. We show genetically that the protein kinase domain is essential for all RAD53-dependent functions tested because a site-specific mutation that inactivates the protein kinase activity results in a mutant phenotype indistinguishable from that of a null mutant. Overexpression of RAD53 , or its kinase domain alone, resulted in a delay in cell-cycle progre ssion that required the intact kinase function. The cell-cycle delay d id not require any of the checkpoint genes tested (e.g. rad9 or mec1), indicating that the cell-cycle delay is either unrelated to the check point responses, or that it occurs constitutively because RAD53 acts f urther downstream of the checkpoint genes tested. Finally, elimination of sequences in the promoter region of RAD53 revealed complex regulat ory elements. (C) 1997 by John Wiley & Sons, Ltd.