Explore the vast range of titles available.

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.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.Non-homologous DNA end joining (NHEJ) is the main repair pathway of DNA double-strand breaks. Recent studies show that synapsis — the crucial pairing of DNA ends — is performed by several mechanisms, and this insight can now be integrated with updates on the DNA end processing and ligation steps of NHEJ, and with NHEJ-related human diseases.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway — alternative end joining (a-EJ) — has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.DNA polymerase theta (Polθ)-mediated end joining is a recently characterized DNA repair pathway that functions in various cellular contexts to repair DNA double-strand breaks that are not repaired by other pathways. Polθ-mediated end joining both helps maintain the genome and causes genome instability, and is an emerging therapeutic target in cancer.

Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming γH2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of γH2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for γH2Ax landscapes. DSBs that disrupt a topological border permit extension of γH2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with γH2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity. Formation of γH2Ax serves as a checkpoint for double-strand break (DSB) repair pathways. Here the authors reveal via integrated chromatin analysis that γH2Ax domains are established by chromosomal contacts with the DSB site.

The targeting range of the CRISPR endonuclease Cpf1 is increased three-fold by molecular engineering. The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells 1 , 2 , 3 , 4 , 5 , 6 , 7 . However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS 7 indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells. CRISPR/Cas9 and transcription activator-like effector nuclease (TALEN) are becoming major tools for genome editing. Here, Song et al . show that RS-1, a small-molecule enhancer for homology directed repair, increases the CRISPR/Cas9 and TALEN mediated knock-in efficiency both in vitro and in vivo with rabbit.

Key Points p53-binding protein 1 (53BP1) is a crucial component of DNA double-strand break (DSB) signalling and repair in mammalian cells. It is recruited to DSBs downstream of RING finger 8 (RNF8)- and RNF168-dependent chromatin ubiquitylation. It reads a DSB-specific histone code that directly integrates ubiquitylation, methylation and acetylation signals at damaged chromatin. Oligomerized 53BP1 binds directly to mono- and dimethylated Lys20 of histone 4 (H4K20me1 and H4K20me2) via its Tudor domain and to RNF168-ubiquitylated H2AK15 via its ubiquitylation-dependent recruitment (UDR) motif. The access of 53BP1 to mono- and dimethylated H4K20 and its recognition of ubiquitylated H2AK15 are modulated through several distinct mechanisms. 53BP1 is a key regulator of DSB repair pathway choice. During G1, it promotes non-homologous end-joining (NHEJ)-mediated DSB repair by antagonizing long-range DNA end-resection, which is essential for DSB repair via homologous recombination. PTIP (PAX transactivation activation domain-interacting protein) and RIF1 (RAP1-interacting factor 1) are 53BP1 effector proteins during DSB repair pathway choice. They bind to ataxia-telangiectasia mutated (ATM)-phosphorylated Ser/Thr-Gln (S/T-Q) sites in the 53BP1 amino terminus. During S–G2, breast cancer 1 (BRCA1) and its interacting partner CtBP-interacting protein (CtIP) counteract 53BP1–RIF1 and 53BP1–PTIP complexes to promote DNA end-resection and thus homologous recombination-mediated DSB repair. Mechanistically, how 53BP1 and its cofactors block resection in G1 and how these activities are counteracted by BRCA1 to enable DSB repair by homologous recombination in S phase remains an open question in the field. The function of 53BP1 in DNA double-strand break repair is multifaceted, and includes mediator and effector roles. New appreciation of how it is recruited to damaged chromatin, and how it exerts control on pathway choice, has cemented the central role of 53BP1 in genome stability maintenance. DNA double-strand break (DSB) signalling and repair is crucial to preserve genomic integrity and maintain cellular homeostasis. p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DSBs that promotes the end-joining of distal DNA ends, which is induced during V(D)J and class switch recombination as well as during the fusion of deprotected telomeres. New insights have been gained into the mechanisms underlying the recruitment of 53BP1 to damaged chromatin and how 53BP1 promotes non-homologous end-joining-mediated DSB repair while preventing homologous recombination. From these studies, a model is emerging in which 53BP1 recruitment requires the direct recognition of a DSB-specific histone code and its influence on pathway choice is mediated by mutual antagonism with breast cancer 1 (BRCA1).

In studies in mammalian cells, polymerase theta (Polθ, also known as POLQ) is identified as the polymerase responsible for non-homologous end joining DNA repair; this DNA repair pathway acts in many tumours when homologous recombination is inactivated and the identification of the polymerase responsible may aid the development of new therapeutic approaches. Polθ involved in alternative DNA repair The error-prone non-homologous end joining (NHEJ) DNA repair pathway is used as an alternative when the error-free homologous recombination pathway is compromised, or is the pathway of choice in some cellular contexts, such as in the immune system. After broken ends are paired via microhomology, NHEJ depends on DNA synthesis, but the identity of the polymerase involved was unclear. Two studies, from the laboratories of Agnel Sfeir and Alan D'Andrea, now implicate the mammalian POLQ gene, encoding the error-prone polymerase Polθ, in this process. Sfeir and colleagues show that upon telomere deprotection, Polθ is needed to prevent alternative end joining at telomeres, and chromosomal translations at non-telomeric sequences. D'Andrea and colleagues focus on the role of Polθ in cancer cells, and show that in a homologous-recombination-deficient background, the absence of Polθ results in a synthetic lethality, suggesting a possible therapeutic approach. Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair 1 . Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms 2 , 3 , 4 , 5 . Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ ) 6 in a pathway required for the repair of DNA double-strand breaks 7 , 8 , 9 , referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway 10 , 11 , 12 , 13 . Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene ( Fancd2 ) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.

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.

Next-generation sequencing technology is used to show that the error-prone polymerase θ (Polθ) is needed to promote alternative non-homologous end joining at telomeres, and during chromosomal translocations, while counteracting homologous recombination; inhibition of Polθ represents a potential therapeutic strategy for tumours that have mutations in homology-directed repair genes. Polθ involved in alternative DNA repair The error-prone non-homologous end joining (NHEJ) DNA repair pathway is used as an alternative when the error-free homologous recombination pathway is compromised, or is the pathway of choice in some cellular contexts, such as in the immune system. After broken ends are paired via microhomology, NHEJ depends on DNA synthesis, but the identity of the polymerase involved was unclear. Two studies, from the laboratories of Agnel Sfeir and Alan D'Andrea, now implicate the mammalian POLQ gene, encoding the error-prone polymerase Polθ, in this process. Sfeir and colleagues show that upon telomere deprotection, Polθ is needed to prevent alternative end joining at telomeres, and chromosomal translations at non-telomeric sequences. D'Andrea and colleagues focus on the role of Polθ in cancer cells, and show that in a homologous-recombination-deficient background, the absence of Polθ results in a synthetic lethality, suggesting a possible therapeutic approach. The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions 1 , 2 , 3 . Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.