Mechanistic Understanding Of PCNA-based Interactions: The Interplay Between CAF-I and Rrm3p for the DNA Sliding Clamp PCNA

Abstract

The Proliferating Cell Nuclear Antigen (PCNA) is a homotrimeric ring that encircles DNA at the replication fork and acts as a docking hub for numerous replication related proteins. Chromatin Assembly Factor I (CAF-I) and rDNA Recombination Mutation 3 (RRM3) have been implicated in the conversions of the epigenetic state during DNA replication and interact with PCNA through a PCNA-Interacting Peptide (PIP) sequence. The exchange of these factors at the replication fork must play a key role in the coordination of successful replication and the maintenance of chromatin structure. I used a co-immunoprecipitation assay and a GST-based pulldown assay to determine the interaction profile of these proteins and observed that a co-operative tri-partite complex seems plausible. Furthermore, I investigated the significance of CDC7 and CDC28 phosphorylation on CAF-I association with PCNA using a Yeast-Two Hybrid assay. My results show that eliminating putative phosphorylation sites reduced association to differing degrees.