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Key Points Mammalian non-homologous DNA end joining (NHEJ) is the primary pathway for the repair of DNA double-strand breaks (DSBs) throughout the cell cycle, including during S and G2 phases. NHEJ relies on the Ku protein to thread onto each broken DNA end. Ku recruits the enzymes and complexes that are needed to trim (nucleases) or to fill in (polymerases) the ends to make them optimally ligatable by the DNA ligase IV complex. The configuration of the DNA ends determines which of several subpathways of NHEJ is able to join the ends. Because NHEJ is flexible and iterative, any of these subpathways can be used but some pathways are more efficient than others for certain DNA ends. When NHEJ is absent owing to a lack of Ku or the DNA ligase complex, alternative end joining (a-EJ) can join the ends using microhomology (usually >4 bp) and there is often some evidence of templated insertions of substantial length (>10 nucleotides). DNA polymerase θ (Pol θ) is of key importance for a-EJ. The single-strand annealing (SSA) pathway requires further end resection by exonuclease 1 (EXO1), Bloom syndrome RecQ-like helicase (BLM) or DNA replication helicase/nuclease 2 (DNA2) to generate the long 3′ single-strand DNA (ssDNA) tails (>20 nucleotides) that are bound by replication protein A (RPA) to prevent the formation of DNA secondary structures. The 3′ ssDNA tails are annealed by RAD52. In mammalian cells, DNA double-strand breaks (DSBs) are repaired predominantly by the non-homologous end joining (NHEJ) pathway, which includes subpathways that can repair different DNA-end configurations. Furthermore, the repair of some DNA-end configurations can be shunted to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA). DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2 -deficient cells from DNA damage and rescues the lethality of Brca2 -deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2 -deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance. Protection of nascent DNA from degradation provides a mechanism that can promote synthetic viability and drug resistance in Brca -deficient cells without restoring homologous recombination at double-strand breaks. Chemoresistance in BRCA cancers The breast cancer susceptibility genes BRCA1 and BRCA2 function to protect the genome from DNA damage. For this reason, DNA-damaging agents are used clinically to treat BRCA -deficient cancers. However, these treatments may have a short window of effectiveness; many cancers develop resistance. André Nussenzweig and colleagues show that cells become drug resistant due to loss of the PTIP protein. In its absence, forks that stall during DNA replication are protected from degradation, and this allows the cells to survive. This work highlights a previously unknown mechanism by which resistance to cancer therapy can arise.

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis 1 , 2 . The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors 2 , 3 . Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans , which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules. Double-strand DNA break repair by the non-homologous end joining pathway involves the transition from a complex that bridges the DNA ends to a complex that aligns the DNA for ligation through the dissociation of the kinase subunits of the DNA-PK complexes.

Genetic studies in yeast indicate that RNA transcripts facilitate homology-directed DNA repair in a manner that is dependent on RAD52. The molecular basis for so-called RNA−DNA repair, however, remains unknown. Using reconstitution assays, we demonstrate that RAD52 directly cooperates with RNA as a sequence-directed ribonucleoprotein complex to promote two related modes of RNA−DNA repair. In a RNA-bridging mechanism, RAD52 assembles recombinant RNA−DNA hybrids that coordinate synapsis and ligation of homologous DNA breaks. In an RNA-templated mechanism, RAD52-mediated RNA−DNA hybrids enable reverse transcription-dependent RNA-to-DNA sequence transfer at DNA breaks that licenses subsequent DNA recombination. Notably, we show that both mechanisms of RNA−DNA repair are promoted by transcription of a homologous DNA template in trans . In summary, these data elucidate how RNA transcripts cooperate with RAD52 to coordinate homology-directed DNA recombination and repair in the absence of a DNA donor, and demonstrate a direct role for transcription in RNA−DNA repair. Homologous recombination (HR) typically uses DNA as a donor template to accurately repair DNA breaks. Here, the authors elucidate two mechanisms by which RAD52 uses RNA as a template for HR: one involving RNA-mediated synapsis of a homologous DNA break, and the other involving reverse transcriptase dependent RNA-to-DNA sequence transfer at DNA breaks.

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.

Metastatic castration-resistant prostate cancer is enriched in DNA damage response (DDR) gene aberrations. The TOPARP-B trial aims to prospectively validate the association between DDR gene aberrations and response to olaparib in metastatic castration-resistant prostate cancer. In this open-label, investigator-initiated, randomised phase 2 trial following a selection (or pick-the-winner) design, we recruited participants from 17 UK hospitals. Men aged 18 years or older with progressing metastatic castration-resistant prostate cancer previously treated with one or two taxane chemotherapy regimens and with an Eastern Cooperative Oncology Group performance status of 2 or less had tumour biopsies tested with targeted sequencing. Patients with DDR gene aberrations were randomly assigned (1:1) by a computer-generated minimisation method, with balancing for circulating tumour cell count at screening, to receive 400 mg or 300 mg olaparib twice daily, given continuously in 4-week cycles until disease progression or unacceptable toxicity. Neither participants nor investigators were masked to dose allocation. The primary endpoint of confirmed response was defined as a composite of all patients presenting with any of the following outcomes: radiological objective response (as assessed by Response Evaluation Criteria in Solid Tumors 1.1), a decrease in prostate-specific antigen (PSA) of 50% or more (PSA50) from baseline, or conversion of circulating tumour cell count (from ≥5 cells per 7·5 mL blood at baseline to <5 cells per 7·5 mL blood). A confirmed response in a consecutive assessment after at least 4 weeks was required for each component. The primary analysis was done in the evaluable population. If at least 19 (43%) of 44 evaluable patients in a dose cohort responded, then the dose cohort would be considered successful. Safety was assessed in all patients who received at least one dose of olaparib. This trial is registered at ClinicalTrials.gov, NCT01682772. Recruitment for the trial has completed and follow-up is ongoing. 711 patients consented for targeted screening between April 1, 2015, and Aug 30, 2018. 161 patients had DDR gene aberrations, 98 of whom were randomly assigned and treated (49 patients for each olaparib dose), with 92 evaluable for the primary endpoint (46 patients for each olaparib dose). Median follow-up was 24·8 months (IQR 16·7–35·9). Confirmed composite response was achieved in 25 (54·3%; 95% CI 39·0–69·1) of 46 evaluable patients in the 400 mg cohort, and 18 (39·1%; 25·1–54·6) of 46 evaluable patients in the 300 mg cohort. Radiological response was achieved in eight (24·2%; 11·1–42·3) of 33 evaluable patients in the 400 mg cohort and six (16·2%; 6·2–32·0) of 37 in the 300 mg cohort; PSA50 response was achieved in 17 (37·0%; 23·2–52·5) of 46 and 13 (30·2%; 17·2–46·1) of 43; and circulating tumour cell count conversion was achieved in 15 (53·6%; 33·9–72·5) of 28 and 13 (48·1%; 28·7–68·1) of 27. The most common grade 3–4 adverse event in both cohorts was anaemia (15 [31%] of 49 patients in the 300 mg cohort and 18 [37%] of 49 in the 400 mg cohort). 19 serious adverse reactions were reported in 13 patients. One death possibly related to treatment (myocardial infarction) occurred after 11 days of treatment in the 300 mg cohort. Olaparib has antitumour activity against metastatic castration-resistant prostate cancer with DDR gene aberrations, supporting the implementation of genomic stratification of metastatic castration-resistant prostate cancer in clinical practice. Cancer Research UK, AstraZeneca, Prostate Cancer UK, the Prostate Cancer Foundation, the Experimental Cancer Medicine Centres Network, and the National Institute for Health Research Biomedical Research Centres.

Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain. CSB in turn is recruited by R loops, which are strongly induced by ROS in transcribed regions. Notably, CSB displays a strong affinity for DNA:RNA hybrids in vitro, suggesting that it is a sensor of ROS-induced R loops. Thus, TC-HR is triggered by R loops, initiated by CSB, and carried out by the CSB-RAD52-RAD51 axis, establishing a BRCA1/2-independent alternative HR pathway protecting the transcribed genome. Transcription-coupled homologous recombination (TC-HR) is activated by reactive oxygen species-induced DNA damage to maintain transcribed genome stability. The authors demonstrate that R loops are induced by ROS at the transcribed genome, triggering a CSB-RAD52- dependent but BRCA1/2-independent RAD51 loading for repair.

Transcription-coupled DNA repair removes bulky DNA lesions from the genome 1 , 2 and protects cells against ultraviolet (UV) irradiation 3 . Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4 CSA and UV-stimulated scaffold protein A (UVSSA) 3 . Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published 3 , 4 data, the structures provide a model for transcription–repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, EC TCR , uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4 CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4 CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA. The authors resolve the structure of five complexes containing RNA polymerase II and the CSA and CSB proteins, offering insight into how the repair of DNA lesions is coupled to transcription.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly 1 , 2 . To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1 , which encodes a crucial DNA repair protein 3 , 4 , in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence 5 – 7 in the immune system only. We show that Vav-iCre +/− ;Ercc1 −/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice 8 – 10 . Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre +/− ;Ercc1 −/fl or aged wild-type mice into young mice induced senescence in trans , whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre +/− ;Ercc1 −/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function 11 , 12 . These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing. An aged, senescent immune system has a causal role in driving systemic ageing, and the targeting of senescent immune cells with senolytic drugs has the potential to suppress morbidities associated with old age.