Screening for the Functionality of RNA Templated Repair of Double-Strand Breaks in Saccharomyces Cerevisae

Abstract

Double-strand breaks (DSBs) in DNA are detrimental, as they can cause mutations and genomic rearrangements, which in turn leads to cancer and other diseases. Recent research reveals that DSBs can be repaired by RNA-templated homologous recombination. However, RNA-templated repair of DSBs is not well understood. In order to better understand the mechanism of RNA-templated repair of DSBs, the current research aims to identify the proteins that facilitate the repair. The research utilizes a system wherein RNA-templated repair of DSBs is known to occur. A yeast overexpression plasmid library was produced in order to test the ability of fragments of the yeast genome to facilitate RNA-templated repair of a DSB when these are highly expressed in the yeast cells. In order to test the ability of added gene fragments to facilitate RNA-templated repair of DSBs, the experimental candidates were exposed to galactose in order to induce a DSB, to activate the transcription of the overexpressed gene fragment, and to initiate the transcription of anti-sense RNA used to repair the break. The candidates were then moved to medium without histidine in order to assess the frequency of repair. Once a large number of colonies (~50,000) are screened, we expect to identify several proteins that facilitate RNA-templated repair of DSBs. Identifying the specific genes that facilitate this repair mechanism will assist in characterizing the functionality of the RNA-templated DNA repair mechanism. Identifying these genes will also allow for better predictions for how this same phenomenon may occur in human cells.