Topoisomerase III acts upstream of Rad53p in the S-Phase DNA damage checkpoint

Deletion of the Saccharomyces cerevisiae TOP3 genes encoding Top3p, leads t o a slow-growth phenotype characterized by an accumulation of cells with a late S/G(2) content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Ar thur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have inves tigated the function of TOP3 during cell cycle progression and the molecula r basis for the cell cycle delay seen in top3 Delta strains. We show that t op3 Delta mutants exhibit a RAD24-dependent delay in the G(2) phase, sugges ting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3 De lta strains are sensitive to killing by a variety of DNA-damaging agents, i ncluding UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phas e checkpoint defect is associated with a defect in phosphorylation of Rad53 p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consi stent with a role for Top3p specifically during S phase, top3 Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G, phase, and DNA damage checkpoints operati ng outside of S phase are unaffected by deletion of TOP3. All of these phen otypic consequences of loss of Top3p function are at least partially suppre ssed by deletion of SGS1, the yeast homologue of the human Bloom's and Wern er's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model p roposing a role for these proteins in S phase is presented.