Abstract 1527 Investigating the Effect of ATG3 Mutations on Autophagy in Yeast for Understanding Autophagosome Number and Size Regulation

Introduction: The study of mutagenesis and DNA repair can lead to the identification of genes and proteins that can be targeted to specifically induce cell death or reduce mutagenesis.The DNA repair proteins that were examined in this study were Rev1, which has a carboxy-terminal domain (CTD) that can recruit error-prone translesion polymerases to DNA lesions; polymerase eta (Polh), which is a translesion polymerase; and CG7139, which is predicted to be a DNA endonuclease.The purpose of this study was to further characterize the role of DNA repair proteins Polh and CG7139 in DNA alkylation damage tolerance in Drosophila melanogaster.Methods: Mutagen sensitivity assays were conducted to assess the sensitivity of single and double mutant larvae to methyl methanesulfonate (MMS), an alkylating agent that causes base mispairings and stalled replication forks.Results: Mild sensitivity to MMS was observed in the single mutants that had null mutations in either Polh or CG7139, and extreme hypersensitivity was observed at moderate and low levels of MMS in the Polh, Rev1DCTD and CG7139, Rev1DCTD double mutants.The drastic difference in sensitivity to MMS between the single and double mutants demonstrates the importance of the Polh and CG7139 proteins in tolerating alkylation damage when translesion synthesis is compromised by the loss of the Rev1-CTD.Conclusion: The hypersensitivity observed in the mutants lacking Polh and the Rev1-CTD supports our model that Polh acts to bypass DNA lesions in post-replication singlestranded gap repair.The hypersensitivity observed in the mutants lacking CG7139 and the Rev1-CTD demonstrate the critical importance of CG7139 in addressing MMS-induced alkylation damage when TLS is compromised.We are currently exploring possible roles of CG7139 as an endonuclease that cleaves DNA intermediates that arise during template switching.