Investigating the role of the mismatch repair proteins MSH2 and MSH6 in genome stability - PROJECT SUMMARY Homologous recombination is a critical pathway for repair of DNA lesions, including double-stranded breaks, ssDNA gaps, and stalled or collapsed replication forks. Of particular importance is the selection of the duplex template for repair. Recombination between non-allelic repeats instead of the sister chromatid can lead to genome rearrangements and loss of heterozygosity, both of which have demonstrated significance to an array of human diseases, particularly cancer. The mismatch repair proteins MSH2 and MSH6 form a heterodimer that detects base-base mismatches and small insertions/deletions. Not only does it function in postreplicative mismatch repair, MSH2-MSH6 has also evolved to prevent recombination between mismatched sequences. Despite the importance of this activity to genome stability, the mechanisms involved remain poorly characterized. In this proposal, I will use a combination of genetic and genomic approaches to delineate the mechanism(s) through which MSH2-MSH6 rejects mismatched recombination intermediates and the define the clinical significance of loss of this important activity in MSH2/6-deficient patient tumors. In Aim 1A (K99 phase), I will use novel proximity ligation-based assays in the model organism Saccharomyces cerevisiae in combination with genetic analysis to delineate the mechanism of MSH2-MSH6 rejection of mismatched recombination intermediates. I will examine recombination between an inducible double-stranded break and each of two templates, one that is perfectly matched, and one that contains ~5% mismatches. I will determine how MSH2-MSH6 biases recombination towards the perfectly matched template and identify the effectors that it recruits to achieve this outcome. In Aim 1B (R00 phase), I will transfer the proximity ligation assays from yeast to human cells and characterize MSH2-MSH6-dependent regulation of mismatched recombination intermediates in this system. In Aim 2, I will use two different approaches to systematically characterize the mutational signatures associated with loss of regulation of recombination by MSH2-MSH6 in MSH2/6-deficient patient tumors as compared to matched healthy tissue controls. In Aim 2A (K99 phase), I will use unbiased long-read whole-genome sequencing and a genome-wide bioinformatic approach to identify de novo structural variants in the patient tumor samples. In Aim 2B (R00 phase), I will use targeted sequencing of long reads containing human lineage-specific LINE-1 elements, which are expected to be disproportionately involved in recombination-mediated rearrangements as a result of their length and the high sequence identity of elements within relatively young LINE-1 subfamilies to one another. This work will provide important insight into the processes that prevent recombination between ectopic repeats and will serve as the foundation of my research program as I transition to an independent faculty career.
