PERSISTENT INITIATION OF DNA-REPLICATION AND CHROMATIN-BOUND MCM PROTEINS DURING THE CELL-CYCLE IN CDC6 MUTANTS

Faithful inheritance of genetic information requires that DNA be copie d only once each cell cycle. Initiation of DNA replication involves th e establishment of a prereplication complex (pre-RC) and subsequent ac tivation by CDK/cyclins, converting the pre-RC to a post-RC. The origi n recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA rep lication, increased nuclear DNA content, and constant MCM protein asso ciation with chromatin throughout the cell cycle. This gain-of-functio n cdc6 mutant ignores the negative controls imposed normally on initia tion by the CDK/cyclins, suggesting that Cdc6p is a key mediator of on ce-per-cell-cycle control of DNA replication.