CHARACTERIZATION OF METAZOAN ORIGINS OF REPLICATION

University Of California San Diego, La Jolla CA

Investigators

Linked publications & trials

Abstract

Origins of replication are characterized in viral, bacterial, and single celled eucaryote as chromatin sites where initiation of replication begins. In metazoans origin recognition complexes (ORC) bind specifically in an ATP dependent manner and initiate the formation of a pre-replicative protein complex composed of ORC, CDC6 and MCM proteins. However, the inability to isolate specific cis acting DNA elements in metazoans has suggested that origins, though conserved in theme, are dependent on other factors. In fact, extracts of Xenopus eggs faithfully execute a single round of cell cycle regulated semi-conservative DNA replication independent of the source of DNA template. This suggests that replication is sequence independent in metazoans and may depend on higher order chromatin or nuclear structure. To characterize metazoan origins in higher organisms I will use a plasmid competition assay to calculate an off-rate for chromatin bound ORC in Xenopus extracts. ORC binding in extracts is markedly affected by both the presence of other replication proteins such as CDC6 and MCM as well as the hydrolysis of ATP. This suggests a testable model of origin selection for metazoans and allows for the determination of sequence and structure dependency in vitro.