DISRUPTION OF RE-REPLICATION CONTROL BY OVEREXPRESSION OF HUMAN ORC1 IN FISSION YEAST

Initiation of DNA replication in Saccharomyces cerevisiae requires the binding of the origin recognition complex (ORC) to autonomously repli cating sequences. Hs-ORC1, a recently identified human protein related to yeast Orc1p and Cdc6p/Cdc18p, may be a component of the replicatio n initiation complex in human cells. We have independently isolated th e gene for HsORC1 and begun to address its function in eukaryotic DNA replication and its relationship to Cdc18p. Although HsORC1 failed to rescue the temperature-sensitive S. pombe cdc18-K46 strain, overexpres sion in a wild-type strain led to continuous DNA synthesis in the abse nce of mitosis. Deletion mutagenesis identified a short N-terminal reg ion of HsORC1 that contains potential phosphorylation sites for cyclin -dependent kinase (CDK) as being sufficient to induce re-replication. In addition, we found that HsORC1 is an efficient substrate for CDKs i n vitro. We propose that perturbation of the re-replication control by overexpression of HsORC1 is due to a titration of components involved in inactivating Cdc18p upon initiation of replication.