Abstract #35

Cell Cycle Control of DNA Replication. Richard Morreale, Brian Green, Muluye Liku, Joachim J. Li. Univ. California, San Francisco, San Francisco, CA.
   We are interested in understanding the regulatory mechanisms that prevent re-replication within a single eukaryotic cell cycle. We have previously shown in Saccharomyces cerevisiae that cyclin dependent kinases (CDKs) prevent re-replication by phosphorylating and down regulating three components of the pre-replicative complex (pre-RC), Mcm2-7, Cdc6 and ORC; simultaneous disruption of these mechanisms is needed to detect significant, but incomplete, re-replication in G2/M phase. To better understand how this deregulation leads to re-replication and why it is incomplete, we have performed a genome-wide analysis of this re-replication using microarray comparative genomic hybridization (CGH). We observe that re-replication is limited to specific regions of the genome for two reasons: (1) re-initiation only occurs at a subset of the origins that are available for S phase initiation and (2) elongation is restrained during re-replication. These data show that there are additional mechanisms blocking re-replication and that origins differ in their susceptibility to re-replication. We have identified one of these mechanisms, and it involves CDK inhibition of Pol12, a subunit of the DNA polymerase alpha-primase complex whose recruitment to origins during S phase initiation is promoted by CDK activation. This is the first example of CDKs targeting a non-pre-RC component to prevent re-initiation and it raises the question of how CDKs can both positively and negatively regulate non-pre-RC factors without generating a mechanistic conflict. We also demonstrate re-replication can indeed lead to genomic instability by inducing gene duplication and possibly gene amplification. Finally, the microarray CGH assay also reveals that deregulation of fewer initiation proteins still leads to re-replication, albeit at levels undetectable by standard replication assays. This finding indicates that the multiple mechanisms preventing re-replication are not strictly redundant and suggests that re-replication may occur more frequently and be a greater source of genome instability than previously appreciated.

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