The Role of SGS1 Helicase in Mismatch Repair During Single Strand Annealing

Abstract

In humans, interspersed repeated sequences constitute at least 35% of the genome. Single Strand Annealing (SSA) is a repair pathway which occurs when a double strand break is produced between homologous repeats. The product of SSA is a single copy of one of the homologous repeats. As such SSA is a highly mutagenic repair pathway. If an SSA event occurs and consequently deletes important genes such as BRAC2, which itself contains repeats, the result can be catastrophic.To study mismatch correction (MMC) during SSA, a yeast strain was created to have 7 mismatches (6 base‐pair substitutions and 1‐bp insertion/deletion), resulting in a 3% divergence between the repeats. Previous results show that strains with 3% divergence between the repeats, designated as the F‐A strain, reduced SSA two‐fold compared to the identical strains, designated as the A‐A strain. This reduction in SSA is due to heteroduplex rejection which occurs when the mismatch repair complex, MSH2‐MSH6, recognizes the mismatches in the DNA‐duplex and stimulates the unwinding of the DNA via SGS1 helicase, resulting in cell death. In the absence of heteroduplex, MSH2‐MSH3 will bind branched SSA intermediate, and recruit RAD1‐RAD10 to remove the non‐homologous tails.Sgs1Δ results in a very efficient SSA repair, with 2 folds increased viability, in both F‐A and A‐A strains, compared to WT strains as SGS1 is required for heteroduplex rejection. Sequencing analysis of sgs1Δ F‐A strain shows an asymmetric gradient of correction favoring the F fragment, similar to the WT strain, suggesting that SGS1 has minimal effects on MMC outcomes. In both sgs1Δ and WT strains tailless strains, there is a uniform correction of MMs, suggesting that the non‐homologous tails influence MMC. All together current data suggests that MMC is more likely to occur during replication, once the SSA repair is completed. Future directions include knocking‐out MLH1, a mismatch repair protein shown to interact with SGS1, and investigating if sectored colonies, cells with unrepaired mismatches is due to replication.Support or Funding InformationBrandeis Division of Science Summer Research Fellowship