RPA Phosphorylation Inhibits DNA Resection

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Bloom’s syndrome (BLM) helicase together with exonuclease 1 (EXO1) and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which further stimulates DNA resection. RPA is phosphorylated during resection as part of the DNA damage response (DDR). Remarkably, pRPA inhibits DNA resection in cellular assays and in vitro via inhibition of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA provides a feedback loop between DNA resection and the DDR. [Display omitted] •Phosphorylated RPA (pRPA) inhibits DNA resection in vitro and in cells•RPA stimulates BLM initiation at DNA ends and inhibits strand-switching activity•BLM helicase stimulates EXO1/DNA2 resection past nucleosomespRPA stalls DNA resection at nucleosomes Termination of DNA resection is critical for preserving genome stability during DNA break repair. Soniat et al. report that phosphorylated RPA is a negative regulator of BLM helicase and DNA resection. As phosphorylated RPA accumulates during resection, this work establishes a negative feedback loop between resection and its termination.