Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase

In Saccharomyces cerevisiae, replication origins are activated with charact eristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) an d Cdc7p-Dbf4p kinase are required far origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, ra ising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that bath Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times, The origin associations of Cdc45p and RPA are mutually dependent, an d both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote load ing of Cdc45p and RPA onto a preformed prereplication complex at each origi n with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 elem ent of ARS1, a potential origin DNA-unwinding element. Cdc45p is required f or recruiting DNA polymerase alpha onto chromatin, and it associates with M cm2p, RPA, and DNA polymerase epsilon only during S phase, These results su ggest that the complex containing Cdc45p, RPA, and MCMs is involved in orig in unwinding and assembly of replication forks at each origin.