Loss control of Mcm5 interaction with chromatin in cdc6-l mutated in CDC-NTP motif

Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and m aintaining the prereplicative complex (pre-RC) by interacting with the orig in recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs ) and other initiator proteins onto replication origins. Although the tempe rature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studie s, the molecular mechanism of the cdc6-1 mutation has been unclear, In this study, we have identified a base substitution at Gly(260)-->Asp, near the CDC-NTP motif, Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins. Chromatin fra ctions were used to study Mcm5 binding in both the wildtype and mutant back ground, These studies indicated that Cdc6 is also involved in unloading Mcm 5 from chromatin. Specifically, the cdc6-1 mutation protein, cdc6(G260D), w hich failed to load Mcm5 onto replication origins, also failed to unload th e Mcm5 protein. Furthermore, the overexpression of wildtype CDC6 accelerate d the unloading of Mcm5 from chromatin fractions. In the absence of functio nal Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the c ell cycle arrested at the G(1)S phase transition. Our results suggested tha t the cdc6(G260D) mutant protein fails to assemble an operational replicati ve complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins.