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Origin-Dependent DNA Replication in vitro: Insights into DDK and S-CDK function and Replisome Assembly

ABSTRACT: Initiation of eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding in vivo studies found that DDK was required for Cdc45 binding at early origins during G1. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of the lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to DNA unwinding and initial RNA primer synthesis. We performed chromatin immunoprecipitation (ChIP) against Cdc45 in CDC7 and cdc7-4 cells arrested in G1 phase to assess the requirement of the Dbf4-dependent kinase on the recruitment of Cdc45 to origin DNA during G1.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Stephen Bell

PROVIDER: E-GEOD-29646 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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