G2 M CHECKPOINT GENES OF SACCHAROMYCES-CEREVISIAE - FURTHER EVIDENCE FOR ROLES IN DNA-REPLICATION AND/OR REPAIR/

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RA D24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong simila rity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis. a 3' to 5' DNA exonuclease/checkpoint prot ein. and the checkpoint protein Rad1p from Schizosaccharomyces pombe ( as we previously reported). Rad24p shows sequence similarity to replic ation factor C (RFC) subunits, and the S. pombe Rad17p checkpoint prot ein, suggesting it has a role in DNA replication and/or repair. This h ypothesis is supported by our genetic experiments which show that over expression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44 ), DNA pola (cdc17) and DNA pol delta (cdc2) but has much weaker effec ts on cdc6, cdc9, cdc15 and CDC+ strains. The idea that RAD24 overexpr ession induces DNA damage, perhaps by interfering with replication/rep air complexes, is further supported by our observation that RAD24 over expression increases mitotic chromosome recombination in CDC+ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly bec ause they are required for the repair of HU-induced DNA damage. In add ition, all three are required for the rapid death of cdc13 rad9 mutant s. All our data are consistent with models in which RAD17, RAD24 and M EC3 are coordinately required for the activity of one or more DNA repa ir pathways that link DNA damage to cell cycle arrest.