The role of D‐loop synthesis and progression in trinucleotide repeat stability via a break induced replication (BIR) model.

Abstract

Homologous recombination (HR) is a complex, multi‐step process, that has been linked to increasing instability of trinucleotide repeats in Saccharomyces cerevisiae. One step of HR, formation and progression of the D‐loop, involves the invasion of ssDNA into a homologous sequence in the genome. Due to the involvement of a variety of polymerases and accessory factors during D‐loop synthesis and progression, it is possible to hypothesize that there is a high risk of slippage and stalling which could lead to changes in length (instability) of a CAG repeat. Break induced replication (BIR) is a genomic repair process that involves the progression of a D‐loop over long tracts of DNA, and thus is a good model to analyze this structure. In this project an assay system, involving a galactose inducible HO double strand break, results in the formation of a D‐loop due to homology to the URA3 gene on both the left and right arms of chromosome V. Repair through this tract of DNA on the right arm of chromosome V has been characterized through measures of viability, PCR of trinucleotide repeat tract, as well as Southern and Western blots. The characterization of this repair currently leads to the ultimate goal of discerning the impact of different DNA repair mutants on tract length changes within the assay system.Support or Funding InformationTufts University, Freudenreich Lab