Analysing genetic interactions to identify genes, that when overactive, impact genome integrity in Saccharomyces cerevisiae

Abstract

Genomic stability is critical to the survival of an organism as the genome and its interaction with the environment, determines an organism's phenotype at a given time. The DNA of organisms is under constant threat of damage from both exogenous and endogenous mutagenic agents and processes. To counter this, cells have many DNA damage repair pathways to repair damage or to protect DNA from damage. When genes that are involved in DNA repair are damaged the organisms' genome can become increasingly unstable and a mutator phenotype can develop as damage can no longer be repaired. Many disorders related to genetics are caused by the loss of gene function due to mutations in the gene or its promoter and so many studies examined knocking out genes to study the gene function however mutations that cause a gene to be expressed at an aberrantly higher level can also be important for genome stability. As increased expression of a gene due to mutations in a promoter, enhancer or repressor region can lead to cellular disfunction and disease. It is therefore equally useful to overexpress genes to understand their function and potential impact on DNA integrity. Though many studies have assessed the effect of knocking out genes on genetic instability, much fewer have looked at overexpressing genes for this purpose. DNA double strand breaks (DSB) are one of the most catastrophic types of DNA damage and are known to lead to the progression of cancer. The aim of this study is to identify genes, that when overexpressed lead to DSB, and if possible, elucidate the mechanism through which they affect genome integrity in the model organism, Saccharomyces cerevisiae. To this end, genome wide overexpression libraries were constructed and screened in a wild-type and DSB repair deficient background, genes identified were overexpressed in a sub-array of DNA related mutants and direct assays for DSB were carried out on genes of interest. This led to the identification of six genes that appeared to lead to increased DSB when overexpressed. The genes identified by this approach were POL30, RNH202, SMT3, POM152, UBX5 and MLH1. POL30 and RNH202 overexpression were previously implicated in increased sister chromatid exchange and chromosome instability respectively, while SMT3, POM152, UBX5 and MLH1 are novel findings.

Date of Award	2019
Original language	English