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eSymposia | Genomic Stability and DNA Repair


Assessing the role of DNA end resection in mediating homologous recombination


Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Description

Assessing the role of DNA end resection in mediating homologous recombination Homologous recombination (HR) is a relatively error-free mode of DNA double strand break (DSB) repair in which a homologous template is used to repair the break. HR requires the nucleolytic degradation of 5’ ends on either side of the break, which typically occurs in a two-step mechanism. In the first step, the Mre11-Rad50-Xrs2 (MRX) complex along with phosphorylated Sae2 initiate resection, creating short 3’ ssDNA tracts. Another set of nucleases (Exo1, Dna2-Sgs1) can then extend these 3’ overhangs in the second step, which can result in ~3-6kb of 3’ ssDNA. However, it has been shown that both mitotic recombination occurs efficiently with as little as 250 bp of homology. These observations call into question the need for extensive resection for HR. Here we use direct repeat and ectopic recombination assays to study the role of extensive resection in mediating recombination from a DSB generated in one of the repeats. Consistent with previous studies, we find that extensive resection is necessary for single strand annealing (SSA), but interestingly extensive resection is dispensable for gene conversion (GC) in the same direct repeat system. However, extensive resection is required for efficient GC in the ectopic system that contains the same repeats. We additionally found that the lack of Mre11 moderately impairs SSA and both direct repeat and ectopic GC. Elimination of Rad9 does not have a significant effect on the frequency of SSA or GC in the direct repeat system, but does change the types of recombinants recovered. Our results suggest that the requirement for extensive resection in mediating ectopic GC is not due to insufficient resection tract lengths since GC occurs at WT levels in the direct repeat assay in the exo1∆ sgs1∆ cells. Extensive resection is necessary for full activation of the DNA damage checkpoint, which may be more important for ectopic repair. Therefore, future work will aim to explore whether the requirement for extensive resection in mediating ectopic GC is due to its role in checkpoint activation.

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