Interaction of the Irc5 chromatin remodeller and the replicative sliding clamp, PCNA (360G-Wellcome-207352_Z_17_Z)

Irc5 is a budding yeast chromatin remodeller that belongs to the same subfamily as the mammalian HELLS protein. Both have been implicated in the response to DNA damage and the HELLS protein is expressed abnormally or mutated in a number of cancers, yet their mechanism of action is not known. Recently, the N-terminus of Irc5 has been shown to interact directly with the replicative sliding clamp PCNA. Many other proteins involved in maintaining genome integrity interact with PCNA, several via a conserved PIP (PCNA-Interacting Peptide) box motif. A sequence similar to known PIP box motifs has been identified within the N-terminal domain of Irc5 and is the basis for this research. The first aim is to determine whether Irc5 interacts with PCNA via PCNA’s IDCL (inter-domain connecting loop), a region that PIP box proteins commonly bind. This will involve mutating two IDCL residues known to disrupt other PIP box- IDCL interactions, cloning, expressing and purifying the mutated protein and testing its ability to interact with Irc5 by pull-down assays. Secondly, Irc5- PCNA interactions will be disrupted in budding yeast using substitutions in the putative PIP-box of Irc5, to determine if Irc5- PCNA interactions impact genetic stability in vivo.