The interactions between Pol30p (PCNA, Proliferating Cell Nuclear Antigen), Chromatin Assembly Factor -1 and Rrm3p: the role of the CDC28 and CDC7 protein kinases
Piriththiv Dhavarasa, Brandon Wyse, Hollie Rowlands, Lauren Fedak, Mathew Guilleman and Krassimir Yankulov
Department of Molecular and Cellular Biology, University of Guelph, Ontario
The destruction of Chromatin Assembly Factor -1 (CAF-1) or the DNA helicase RRM3 affect the frequency of epigenetic conversions at the telomeres of S.cerevisiae. CAF-I is a histone chaperone, which reassembles nucleosomes from old and new histones immediately after the passage of the replication fork. RRM3 is a helicase that relieves replication forks that have paused at sites of tight protein binding or active transcription. Both Rrm3p and CAF-1 interact with Pol30p, the yeast homologue of the Proliferating Cell Nuclear Antigen (PCNA). These interactions are mediated by a dedicated conserved PCNA-Interacting-Peptide (PIP) consensus sequence present on both the largest subunit of CAF-1 (Cac1p) and on Rrm3p. Interestingly, there are many other proteins that interact with PCNA via PIPs thus suggesting an intriguing interplay of PCNA-binding factors at the replication fork. At the same time, the interaction between CAF-1 and PCNA is in part regulated by two Cyclin-Dependent Kinases, CDC28 and CDC7.In an attempt to mechanistically decipher the effects of CAF-1 and Rrm3p on epigenetic conversions, in this poster we address two important issues:
The possibility of competition and/or cooperating between Pol30p, Cac1p, Rrm3p and several other PCNA-binding proteins.
The extent to which protein kinases regulate the stability of CAF-I and its association with PCNA.
We will show evidence from extensive competitive and non-competitive two-hybrid assays with POL30, CAC1 and RRM3. The effect of mutations on multiple phosphorylation sites of Cac1p on the stability of CAF-I and its ability to associate with Pol30p in cell extracts will also be presented. Finally, we will show our analysis with recombinant proteins on the competition between Cac1p and Rrm3 for their interaction with Pol30p.