Identification of PCNA Binding Sites on Chromatin Assembly Factor 1

Proper organization and maintenance of genomic DNA are important for human health. Immediately after replication, newly synthesized DNA is organized into structures called nucleosomes. Nucleosomes make up the basic structure of chromatin, and their relative compaction influences the level of expression of genes within a particular region of DNA. This selective gene expression is necessary for cells to grow and adapt to changes in their environments. As a result, it is important that the level of DNA compaction is preserved through DNA replication. The process of replication-coupled nucleosome assembly is mediated by two key proteins: chromatin assembly factor 1 (CAF-1), the heterotrimeric protein complex that deposits histone proteins onto newly synthesized DNA, and proliferating cell nuclear antigen (PCNA), a homotrimeric sliding clamp that recruits and regulates proteins during replication and repair. However, how these two proteins interact and function together to couple replication to nucleosome organization is not well understood. In yeast, CAF-1 is composed of three subunits - Cac1, Cac2, and Cac3 - and studies have shown that CAF-1 interacts with PCNA via the largest subunit, Cac1. In particular, PCNA is predicted to interact with Cac1 via a PCNA interacting peptide (PIP) motif. Here, we used enzyme-linked Immunosorbent assays and other in vitro protein-protein binding experiments to identify new functional motifs within Cac1 that interact with PCNA. We are currently carrying out mutational analysis of these motifs to map the specific residues involved in the CAF-1-PCNA interaction.