CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

Summary BLM or WRN helicases function with the DNA2 helicase-nuclease to resect DNA doublestrand breaks and initiate homologous recombination. Upon DNA unwinding by BLM/WRN, RPA directs the DNA2 nuclease to degrade the 5’-strand, revealing the 3’ overhang needed for recombination. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single molecule biochemistry, we show that phosphorylated CtIP dramatically stimulates the ATP hydrolysis driven motor activity of DNA2. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. The domains of CtIP required to stimulate DNA2 are separable from those that regulate the MRN complex. These results establish that CtIP couples both MRE11-dependent short and DNA2-dependent long-range resection, and show how the motor activity of DNA2 promotes resection. Our data explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP. Highlights Phosphorylated CtIP stimulates the motor activity of DNA2 The activated DNA2 translocase facilitates degradation of RPA-coated ssDNA CtIP promotes both MRN and DNA2 nucleases coupling short and long-range resection The CtIP domains required to promote DNA2 and MRN are distinct and fully separable