53BP1-shieldin-dependent DSB processing in BRCA1-deficient cells requires CST-Polα-primase fill-in synthesis

Mirman et al. report that the primary function of the shieldin complex in double-strand break repair in BRCA1-deficient cells is the recruitment of the CST-Pol alpha-primase complex to conduct fill-in synthesis. The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Pol alpha-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Pol alpha) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Pol alpha-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1 Delta) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1 Delta to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1 Delta was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Pol alpha-primase is the major effector of shieldin-dependent DSB processing.

Automated summary

The primary function of the shieldin complex in double-strand break repair in BRCA1-deficient cells is to recruit the CST-Pol alpha-primase complex for fill-in synthesis. The effectiveness of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which limit single-stranded DNA at double-strand breaks through blocking resection and/or fill-in by CST-Pol alpha-primase. CST-Pol alpha-primase promotes radial chromosome formation and BrdU incorporation at DSBs in PARPi-treated BRCA1-deficient cells, and shieldin acts primarily by recruiting CST for radial formation.