Characterization of the checkpoint gene RAD53/MEC2 in Saccharomyces cerevisiae

Sangkyu Kim, Ted A. Weinert

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Saccharomyces cerevisiae cells carrying mutations in RAD53/MEC2 fail to arrest in the S phase when DNA replication is blocked (the S/M checkpoint) or in the G2 phase when DNA is damaged (the G2/M checkpoint). We isolated and determined the DNA sequence of RAD53 and found that it is identical to the SPKI gene previously identified by Stern et. al. (1991). In addition to its checkpoint functions, we show here that RAD53 is essential for cell viability because null mutants are inviable. Weak 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 progression that required the intact kinase function. The cell-cycle delay did not require any of the checkpoint genes tested (e.g. rad9 or mec 1), indicating that the cell-cycle delay is either unrelated to the checkpoint responses, or that it occurs constitutively because RAD53 acts further downstream of the checkpoint genes tested. Finally, elimination of sequences in the promoter region of RAD53 revealed complex regulatory elements.

Original languageEnglish (US)
Pages (from-to)735-745
Number of pages11
JournalYeast
Volume13
Issue number8
DOIs
StatePublished - Jun 30 1997

Keywords

  • Cell cycle
  • Checkpoint
  • DNA damage
  • Protein kinase
  • RAD53
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Genetics

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