Cell Cycle Regulation of Mcm Localization

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DESCRIPTION (provided by applicant): We are using the budding yeast Saccharomyces cerevisiae to understand how eukaryotic cells limit DNA replication to one round per cell cycle. Disruption of this regulation can lead to genomic instability, one of the hallmarks of cancer. One conserved mechanism to prevent re-replication is to exclude initiation I proteins from the nucleus after replication initiation has occurred. In budding yeast, nuclear exclusion of the heterohexameric Mcm2-7 replication complex occurs through regulation of Mcm nucleocytoplasmic transport by the cyclin-dependent kinase Cdc28. We have identified nuclear localization signals (NLSs) on Mcm2 and Mcm3 and a potential nuclear export signal (NES) on Mcm3. We have also shown that Cdc28 phosphorylation of Mcm3 near its NLS and NES promotes the net nuclear export of the Mcm2-7 complex. This phosphorylation could work by inhibiting nuclear import, stimulating nuclear export, or doing both. To investigate these possibilities, I will pursue the following specific aims: Aim 1: Determine whether and how Cdc28 regulates Mcm nuclear import Aim 2: Determine whether and how Cdc28 regulates Mcm nuclear export