BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

Repairing DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nano-manipulation allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and addition of PAXX extends this lifetime to ~2s. Further addition of XRCC4, XLF and Ligase IV resulted in minute-scale synapsis and led to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kCal/mol. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.

XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF-XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm) resolution crystal structure of XLF-XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)-MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.

SummaryBRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.