Eukaryotic replication is tightly regulated during the cell cycle. Each replication origin fires once and only once per S phase for the stable propagation of genetic information....[ Read more ]

Eukaryotic replication is tightly regulated during the cell cycle. Each replication origin fires once and only once per S phase for the stable propagation of genetic information.

Initiation events at origins include two fundamental stages: the assembly of pre-replicative complexes (pre-RC) and the triggering of new DNA synthesis. The assembly of pre-RC involves the participation of a number of proteins: the binding of ORC (origin recognition complex) to replication origin, then the recruitment of Cdc6p, Mcm2p-Mcm7p (minichromosome maintenance), as well as Cdc45p. In turn, Cdc7p/ Dbf4p and Cdc28p/ Clbs protein kinases act on the pre-RC to initiate DNA synthesis. Working in series or in parallel, the protein kinases as well as initiation proteins ensure that DNA replication occurs only once in every cell cycle.

However, these proteins are not sufficient for the initiation of DNA replication. I identify a new essential regulator of replication--Noc3p in yeast by a genetic screen for multicopy suppressors of mcm5-1.

Noc3p was shown to bind constitutively to the chromatin at a constant level throughout the cell cycle, and Noc3p is localized to the replication origin, not non-origin sequences on the chromatin by CHIP (chromatin immunoprecipitation) assay. Noc3p physically interacts with Mcm proteins in co-immunoprecipitation experiments. Furthermore, functional analyses of mutant Noc3p demonstrate that the association of Noc3p is indispensable for the loading of Cdc6p onto the chromatin, as well as the recruitment and maintenance of Mcm proteins to chromatin. In addition, mutant cells of noc3 display high loss of plasmids with only one replication origin, and the mutant is defective in entry into S phase. These results suggest that Noc3p is a component of the pre-RC and is directly involved in the regulation of the initiation of DNA replication at origins.