Analysis of the meiotic events that cause Exo1 phosphorylation and the phenotypic significance of this PTM (360G-Wellcome-207127_Z_17_Z)

The conserved protein Exo1 is an important endonuclease/exonuclease required in the DNA damage response for 5’ to 3' end resection, and during crossover designation in meiosis. The host laboratory has discovered a novel post translational modification (PTM) in Exo1 that arrises following Spo11-DSB during yeast meiosis. This summer research project will determine which steps in the meiotic recombination pathway lead to Exo1 phosphorylation and investigate which Kinase catalyses the PTM. I will also contribute to determining which amino acid is phosphorylated, so in the longer term, non-phosphorylatable mutants can be made to understand how the PTM affects DSB repair during meiosis. Whole cell extracts of mutant yeast strains that either cannot progress through the DNA repair pathway or lack a kinase activity will be tested for the presence/absence of phosphorylated Exo1 periodically as the cells undergo meiosis. I will also prepare meiotic cultures for protein extraction and enrichment of PK tagged Exo1 for Mass Spectrometry analysis to map the phosphorylation event. I will use various cell culture and biochemical techniques like protein extraction and quantification, western blots and immunoprecipitation. This yeast model will develop understanding of Exo1 function in higher eukaryotes as yeast Exo1 is homologous to the human Exo1.