The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 28 CRISPR/Cas9 screens against 25 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 840 genes whose loss causes either sensitivity or resistance to DNA damaging agents. Mining this dataset, we uncovered that ERCC6L2, which is mutated in a bone-marrow failure syndrome, codes for a canonical non-homologous end-joining pathway factor; that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

53BP1 with its downstream proteins, RIF1, PTIP and REV7, antagonizes BRCA1-dependent homologous recombination (HR) and promotes non-homologous end joining (NHEJ) in an unclear manner. Here we show that REV7 forms a complex with two proteins, FAM35A and C20ORF196. We demonstrate that FAM35A preferentially binds single-strand DNA (ssDNA) in vitro, and is recruited to DSBs as a complex with C20ORF196 and REV7 downstream of RIF1 in vivo. Epistasis analysis shows that both proteins act in the same pathway as RIF1 in NHEJ. The defects in HR pathway to repair DSBs and the reduction in resection of broken DNA ends in BRCA1-mutant cells can be largely suppressed by inactivating FAM35A or C20ORF196, indicating that FAM35A and C20ORF196 prevent end resection in these cells. Together, our data identified a REV7–FAM35A–C20ORF196 complex that binds and protects broken DNA ends to promote the NHEJ pathway for DSB repair.

The replication protein A (RPA) complex binds singlestranded DNA generated at stalled replication forks and recruits other DNA repair proteins to promote recovery of these forks. Here, we identify Ewing tumor-associated antigen 1 (ETAA1), which has been linked to susceptibility to pancreatic cancer, as a new repair protein that is recruited to stalled forks by RPA. We demonstrate that ETAA1 interacts with RPA through two regions, each of which resembles two previously identified RPA-binding domains, RPA70N-binding motif and RPA32C-binding motif, respectively. In response to replication stress, ETAA1 is recruited to stalled forks where it colocalizes with RPA, and this recruitment is diminished when RPA is depleted. Notably, inactivation of the ETAA1 gene increases the collapse level of the stalled replication forks and decreases the recovery efficiency of these forks. Moreover, epistasis analysis shows that ETAA1 stabilizes stalled replication forks in an ataxia telangiectasia and Rad3-related protein (ATR)-independent manner. Thus, our results reveal that ETAA1 is a novel RPA-interacting protein that promotes restart of stalled replication forks.The faithful replication of DNA is essential for the maintenance of genomic stability and the prevention of cancerpromoting mutations. Replication forks can be stalled by numerous obstacles on the DNA template, including unrepaired DNA damage, DNA-bound proteins, and secondary structures (1). Stalled replication forks are able to restart once the obstacles are removed or become broken (collapse) into DNA double strand breaks, which pose the most serious threat to genome integrity when fork protection fails (2, 3). However, how stalled replication forks are protected is not well understood.The replication protein A (RPA) 3 complex, which consists of RPA1 (RPA70), RPA2 (RPA32), and RPA3 (RPA14), plays crucial roles in a variety of DNA metabolic pathways, including DNA replication, recombination, repair, and DNA damage checkpoint (4 -6). When replication forks stalled, singlestranded DNA (ssDNA) is generated and extended by minichromosome maintenance protein complex helicases (7,8). The ssDNA is bound by RPA, which protects ssDNA from cleavage by nucleases and recruits repair proteins to initiate DNA damage responses. The RPA-ssDNA complex recruits and activates ATR/ATRIP thereby eliciting checkpoint signaling (9). In addition, RPA-ssDNA complex also recruits factors necessary for the stabilization and resumption of stalled replication forks, such as RAD51 (10, 11) and SMARCAL1 (12-15). Recently, several studies have also revealed a physical and functional interaction between RPA and the ubiquitin E3 ligases RFWD3 (16 -18) and PRP19 (19), which ubiquitinate RPA and facilitate replication fork restart. Here, we identified a new RPA interaction protein, ETAA1, whose gene variation has been associated with susceptibility to pancreatic cancer (20). ETAA1 is recruited to stalled replication forks in response to replication stress, and the disruption of ETAA1 leads to fork colla...

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identify a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.