Srs2 functions needed to replicate CAG/CTG hairpins and prevent repeat instability

Abstract

Trinucleotide repeats form secondary DNA structures that can lead to chromosomal instability, breakage, and fork stalling during replication. Srs2, a non‐replicative helicase, anti‐recombinase that can displace Rad51, and interacts with PCNA, is required for reversed fork formation, which facilitates replication through these structures and prevents trinuleotide repeat expansion. However, how exactly Srs2 contributes to fork reversal and repeat stability still remains elusive. To approach this problem, we tested Srs2 mutants deficient in specific domains, which allowed us to investigate the uncoupling of each domain and how each individually contributes to maintaining genome integrity. We tested these mutants with a PCR based stability assay and fragility assay. We show that Srs2 unwinding and Rad51 displacement activities are all required to prevent CAG/CTG repeat expansions, contractions, and chromosomal breakage. We also show that its PCNA interacting domain is critical in preventing chromosomal breakage but not required to prevent repeat instability or fork reversal. Together, these results suggest that there is an uncoupling of Srs2 domain requirement in genome maintenance. Analysis by 2D electrophoresis will further shed light on which activities contribute to fork reversal. This research was funded by National Institutes of Health grant GM063066 and Tufts Department of Biology