ADDITION OF EXTRA ORIGINS OF REPLICATION TO A MINICHROMOSOME SUPPRESSES ITS MITOTIC LOSS IN CDC6 AND CDC14 MUTANTS OF SACCHAROMYCES-CEREVISIAE

Many cell division cycle (cdc) mutants of Saccharomyces cerevisiae exh ibit elevated mitotic loss of pDK243, a 14-kilobase minichromosome wit h a centromere and one autonomous replicating sequence (ARS). Tandem c opies of different ARSs were added to pDK243. The addition of these AR S clusters to pDK243 had no effect on its mitotic loss in cdc7 (protei n kinase), cdc9 (DNA ligase), or cdc16 or cdc17 (DNA polymerase) mutan ts. However, in cdc6 and cdc14 mutants, the mitotic loss of pDK243 wit h an ARS cluster was suppressed by a factor of 6-8 compared to pDK243 without the cluster. This suppression was dependent upon the number of ARSs in the cluster and the integrity of the ARS consensus sequence i n each ARS of the cluster. ARSs are known to be DNA replication origin s. Therefore, the suppression of mini-chromosome loss by ARSs in cdc6 and cdc14 mutants suggests that these mutants are defective in the ini tiation of DNA replication. Since the CDC6 protein appears to act at t he G1/S phase transition, the CDC6 protein may be a factor required at the beginning of S phase to initiate DNA replication at origins. In c ontrast, the CDC14 protein acts after mitosis. We suggest that the CDC 14 protein performs a function late in the cell cycle that may be requ ired for efficient initiation of DNA replication during S phase of the next cell cycle.