COORDINATING DNA-REPLICATION TO PRODUCE ONE COPY OF THE GENOME REQUIRES GENES THAT ACT IN UBIQUITIN METABOLISM

We have developed a genetic screen of the yeast Saccharomyces cel cere visiae to identify genes that act to coordinate DNA replication so tha t each part of the genome is copied exactly once per cell cycle. A mut ant was recovered in this screen that accumulates aberrantly high DNA contents but does not complete a second round of synthesis. The mutati on principally responsible for this phenotype is in the DOA4 gene, whi ch encodes a ubiquitin hydrolase, one of several yeast genes that enco de enzymes that can remove the signalling polypeptide ubiquitin from i ts covalently linked conjugated forms, DOA4 is nonessential, and delet ing this gene causes uncoordinated replication. Overreplication does n ot occur in cells with limiting amounts of Cdc7 protein kinase, sugges ting that entry into S phase is required for this phenotype, The DNA f ormed in doa4 mutants is not highly unusual in the sense that mitotic recombination rates are normal, implying that a high level of repair i s not induced, The temperature sensitivity of doa4 mutations is partia lly suppressed by extra copies of the polyubiquitin gene UB14, but ove rreplication still occurs in the presence of this suppressor, Mutation s in DOA4 cause loss of the free ubiquitin pool in cells under heat st ress conditions, and extra copies of UB14 restore this pool without re storing coordination of replication, We conclude that a ubiquitin-medi ated signalling event directly involving the ubiquitin hydrolase encod ed by DOA4 is needed in S, cerevisiae to prevent uncoordinated DNA rep lication.