UCSF

CDK Regulated Localization of Replication Proteins

Muluye E. Liku

Abstract

The precise inheritance of genetic material requires that eukaryotic cells initiate DNA replication in a highly regulated manner. To prevent any inappropriate re-replication of the genome, initiation at each of the hundreds to thousands of replication origins must occur only once per cell cycle. In Saccharomyces cerevisiae, cyclin dependent kinases have a critical role in inhibiting reinitiation, and must do so without interfering with their role in promoting initiation. Previously, we had shown that CDKs inhibit multiple proteins involved in an early step of replication initiation, i.e. assembly of the pre-replicative complex (pre-RC), so as to prevent re-initiation of DNA replication. Specifically, we had demonstrated that making ORC, Cdc6, and Mcm2-7 refractory to CDK inhibition promotes limited reinitiation and re-replication from some origins. Despite this finding, our understanding of the mechanisms used by CDKs to prevent re-initiation are incomplete.

This thesis presents two different studies that expand our understanding of these mechanisms. In the first part, we acquired a more in depth understanding of how CDKs inhibit Mcm2-7 activity by promoting their nuclear export. We identified key transport regulatory modules on Mcm2 and Mcm3 that control the import and export of the Mcm2-7 complex and showed that phosphorylation of the Mcm3 module alters the balance between the two events so as to favor export. We showed that this Mcm3 module and its CDK regulation evolved quite recently in a budding yeast lineage that includes Saccharomyces cerevisiae and closely related yeast. A more detailed analysis of CDK phosphorylation consensus sites on Mcm3 and other pre-RC components showed that although the precise number and position of sites is not conserved, the clustering of these sites in specific regions is.

Our second study on the CDK inhibition of reinitiation was motivated by the limited re-replication that we observed when we deregulated ORC, Cdc6, and Mcm2-7. This result suggests that CDKs target additional replication components to prevent re-initiation, and we investigated the possibility that DNA polymerase alpha primase, specifically its Pol1 and Pol12 subunits, might be one of these components. We showed that the CDK phosphorylation of Pol1 and Pol12, which appears to be conserved through humans, are unlikely to be activating events that trigger replication initiation, but are in fact delayed till after replication is mostly complete, consistent with a late inhibitory role. We also showed that CDK phosphorylation of Pol1 is required to promote the relocalization of Pol alpha primase from nucleoplasm to nuclear periphery in G2/M phases. Because of these suggestive hints, although we could not establish a role for Pol 1 or Pol12 phosphorylation in the block to re-replication, we suspect that such a role may be uncovered with further investigation.