Eukaryotic DNA replication is necessary for cell division. To initiate replication, three proteins, ORC, Cdc6 and Cdt1, co-assemble into a condensed liquid phase on chromosome surfaces to load the replicative helicase onto DNA. CDK•Cyc-dependent initiator phosphorylation regulates this process to ensure that it occurs once and only once per division cycle; mis-regulation leads to genomic instability, a hallmark of many cancers. Although aberrant CDK•Cyc activity is common in cancer, and regulation of replication initiation is necessary for genomic health, how these pathways conspire to drive genomic instability in cancer remain...

Read More

DNA replication is necessary for cell division. To initiate replication, three proteins, ORC, Cdc6 and Cdt1, co-assemble into a condensed liquid phase on chromosome surfaces to load the replicative helicase onto DNA. CDK•Cyc-dependent initiator phosphorylation regulates this process to ensure that it occurs once and only once per division cycle; mis-regulation leads to genomic instability, a hallmark of many cancers. Although aberrant CDK•Cyc activity is common in cancer, and regulation of replication initiation is necessary for genomic health, how these pathways conspire to drive genomic instability in cancer remains unknown.

This proposal aims to:

1) Understand the molecular language of initiator liquid-liquid phase separation (LLPS)Replication initiator factors have intrinsically disordered regions (IDRs) that drive DNA-dependent LLPS but the mechanism underlying this is unknown. We will:

• Identify the molecular determinants of IDR-DNA and IDR-IDR binding.
• Clarify the role of IDR composition versus linear motifs in LLPS

2) Determine the effect of reversible phosphorylation on initiator action Phosphorylation of initiator IDRs inhibits initiator condensation on chromatin; however, the factors required for this remain unknown. We will:

• Determine the kinase(s) and phosphatase(s) necessary for reversible phosphorylation.
• Examine whether discrete IDR phosphorylation sites differentially affect function

3). Identify changes in initiator spatiotemporal dynamics under pathological conditions CDK. Cyc phosphorylation is the major regulatory pathway targeting initiators and is mis-regulated in cancer; however, how initiator activity goes awry in cancer has not been investigated. We will:

• Assess initiator spatiotemporal dynamics in normal and transformed cells
• Determine if initiator mis-regulation leads to genomic instability.

Read Less