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Loss of functional BRCA1 protein leads to defects in DNA double-strand break (DSB) repair by homologous recombination (HR) and renders cells hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibitors used to treat BRCA1/2-deficient cancers. However, upon chronic treatment of BRCA1-mutant cells with PARP inhibitors, resistant clones can arise via several mechanisms, including loss of 53BP1 or its downstream co-factors. Defects in the 53BP1 axis partially restore the ability of a BRCA1-deficient cell to form RAD51 filaments at resected DSBs in a PALB2- and BRCA2-dependent manner, and thereby repair DSBs by HR. Here we show that depleting 53BP1 in BRCA1-null cells restores PALB2 accrual at resected DSBs. Moreover, we demonstrate that PALB2 DSB recruitment in BRCA1/53BP1-deficient cells is mediated by an interaction between PALB2’s chromatin associated motif (ChAM) and the nucleosome acidic patch region, which in 53BP1-expressing cells is bound by 53BP1’s ubiquitin-directed recruitment (UDR) domain. Although loss of BRCA1 leads to defects in DNA double-strand break repair by homologous recombination (HR) and renders cells hypersensitive to PARP inhibitors, resistance to the drugs can arise. Here the authors reveal that PALB2 chromatin recruitment restores HR in BRCA1-deficient cells depleted of 53BP1.

SUMO modification and the response to DNA damage The occurrence of a double-strand break in DNA activates a complex series of events that recruit to the break many proteins involved in its repair. A number of these proteins are modified by addition of a small protein, SUMO; this modification is performed SUMO ligases. In this work, Jackson and colleagues show that two such ligases, PIAS1 and PIAS4, add various SUMOs onto DNA repair proteins at double-strand breaks. The PIAS ligases are recruited via their SAP domains, and their activity is required for effective repair. SUMOylation by PIAS1 and PIAS4 is also necessary for the further modification of certain repair factors by ubiquitin, a somewhat larger protein adduct related to SUMO. The successive SUMOylation and ubiquitylation of repair proteins regulates their targeting to, and repair of, DNA breaks. Following the formation of a DNA double-strand break (DSB), cells activate the DNA-damage response and recruit a number of proteins to the lesion. Some of these proteins are modified by the attachment of small ubiquitin-related modifier (SUMO). Here, SUMO1, SUMO2 and SUMO3 are shown to accumulate at DSB sites in mammalian cells. SUMO1 and SUMO2/3 accrual requires the E3 ligase enzymes PIAS4 and PIAS1, which promote DSB repair. DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair 1 , 2 . Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions 3 , 4 , 5 , 6 . Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage 7 , 8 , 9 , 10 , 11 . These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.

ATR plays key roles in cellular responses to DNA damage and replication stress, a pervasive feature of cancer cells. ATR inhibitors (ATRi) are in clinical development for treating various cancers, including those with high replication stress, such as is elicited by ARID1A deficiency, but the cellular mechanisms that determine ATRi efficacy in such backgrounds are unclear. Here, we have conducted unbiased genome-scale CRISPR screens in ARID1A -deficient and proficient cells treated with ATRi. We found that loss of transcription factor KLF5 has severe negative impact on fitness of ARID1A -deficient cells while hypersensitising ARID1A -proficient cells to ATRi. KLF5 loss induced replication stress, DNA damage, increased DNA-RNA hybrid formation, and genomic instability upon ATR inhibition. Mechanistically, we show that KLF5 protects cells from replication stress, at least in part through regulating BRD4 recruitment to chromatin. Overall, our work identifies KLF5 as a potential target for eradicating ARID1A -deficient cancers. ATR inhibitors are being developed for treating cancers, but mechanisms that determine their efficacy are unclear. Here, the authors show that transcription factor KLF5 loss sensitizes cells to ATR inhibition through regulating BRD4 chromatin recruitment. This work also identifies KLF5 as a potential target for treating ARID1A -deficient cancers.

Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX. MDC1 and H2AX interact and accumulate at sites of DNA damage, functioning to recruit additional factors involved in the repair process. Here the authors uncover a function for MDC1 that is independent of the presence of H2AX and is mediated through its PST-repeat region.

The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nu-cleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status.

A recent study published in Science reveals the mechanism and biological importance of DNA damage response abrogation in mitotic cells.A recent study published in Science reveals the mechanism and biological importance of DNA damage response abrogation in mitotic cells.

Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (HIS3 and TRP3), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated SPT7 resulted in derepressed HIS3 transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation.

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. Through CRISPR–Cas9 screen, Dev et al. identified that SHLD1/2 inhibition contributes to PARP-inhibitor resistance. Mechanistically, SHLDs promote non-homologous end-joining and antagonize homologous recombination.

CtIP helps maintain genomic stability by promoting DNA double-strand-break repair. Structural and biophysical analyses now show that the N terminus of human CtIP forms a tetrameric structure that is required for resection of broken DNA ends to permit their repair by homologous recombination. Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro . Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.