RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments
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RECQ5 mutation and overexpression have both been associated with human cancer. RECQ5 has been implicated in repair of oxidative DNA damage, a critical pathway in which inherent redundancies may mask a key role for any single factor. Oxidative damage creates DNA nicks. By using CRISPR/Cas9 or CRISPR/Cas9D10A to target double-strand breaks (DSBs) or nicks to specific sites in the human genome our laboratory has shown that nicks can initiate homology directed repair (HDR) by an alternative pathway that is distinct from HDR at DSBs and that efficiently uses single-stranded DNA donors. This alternative pathway is normally inhibited by RAD51, to prevent genomic instability at nicks. To determine the functions of RECQ5 in HDR, we have assayed the effect of its depletion or overexpression at targeted nicks and DSBs. We found that depletion of RECQ5 inhibited HDR at both nicks and DSBs, by either single-stranded or duplex DNA donors. Conversely, overexpression of RECQ5 inhibited HDR at DSBs and HDR by dsDNA donors at nicks, but stimulated HDR by ssDNA donors at nicks. While RECQ5 associates with the moving transcription apparatus, we did not find that these activities of RECQ5 depended upon transcription of the target gene for recombination, or were affected by deletion of the domain of RECQ5 that interacts with RNA polymerase 2. Structure-function analysis did show that stimulation of HDR depended on the RECQ5 helicase ATPase activity, and the ability of RECQ5 to interact with RAD51. None of the effects of RECQ5 depletion or overexpression was evident in cells in which RAD51 filament formation had been inhibited by treatment with siRAD51 or siBRCA2. We conclude that RECQ5 normally supports canonical HDR at both nicks and DSBs, to promote genomic stability. Somewhat paradoxically, RECQ5 overexpression has the unanticipated consequence of promoting genomic instability, apparently overriding the normally suppressive effect of RAD51 to enable nicks to initiate HDR. These results explain the genomic instability associated with both RECQ5 mutations and overexpression.
- Biological chemistry