53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in.

Abstract

Resection of double-strand breaks (DSBs) dictates the choice between Homology-Directed Repair (HDR), which requires a 3′ overhang, and classical Non-Homologous End Joining (c-NHEJ), which can join unresected ends1,2. BRCA1 mutant cancers show minimal DSB resection, rendering them HDR deficient and sensitive to PARP1 inhibitors (PARPi)3–8. When BRCA1 is absent, DSB resection is thought to be prevented by 53BP1, Rif1, and the Rev7/Shld1/Shld2/Shld3 (Shieldin) complex and loss of these factors diminishes PARPi sensitivity4,6–9. Here we address the mechanism by which 53BP1/Rif1/Shieldin regulate the generation of recombinogenic 3′ overhangs. We report that CST (Ctc1, Stn1, Ten110), an RPA-like complex that functions as a Polymeraseα/primase accessory factor11 is a downstream effector in the 53BP1 pathway. CST interacts with Shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and Shieldin-dependent manner. Like loss of 53BP1/Rif1/Shieldin, CST depletion leads to increased resection. Furthermore, in BRCA1-deficient cells, CST blocks Rad51 loading and promotes PARPi efficacy. Finally, Polα inhibition diminishes the effect of PARPi in BRCA1-deficient cells. These data suggest that CST/Polα-mediated fill-in contributes to the control of DSB repair by 53BP1, Rif1, and Shieldin.