Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway.

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing.

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection.

Double-stranded DNA break repair by homologous recombination is initiated by resection of free DNA ends to produce a 3'-ssDNA overhang. In bacteria, this reaction is catalyzed by helicase-nuclease complexes such as AddAB in a manner regulated by specific recombination hotspot sequences called Crossover hotspot instigator (Chi). We have used magnetic tweezers to investigate the dynamics of AddAB translocation and hotspot scanning during double-stranded DNA break resection. AddAB was prone to stochastic pausing due to transient recognition of Chi-like sequences, unveiling an antagonistic relationship between DNA translocation and sequence-specific DNA recognition. Pauses at bona fide Chi sequences were longer, were nonexponentially distributed, and resulted in an altered velocity upon restart of translocation downstream of Chi. We propose a model for the recognition of Chi sequences to explain the origin of pausing during failed and successful hotspot recognition.

Holding it together: DNA end synapsis during non-homologous end joining.

DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.

The high toxicity of DSB-clusters modelling high-LET-DNA damage derives from inhibition of c-NHEJ and promotion of alt-EJ and SSA despite increases in HR.

Heavy-ion radiotherapy utilizing high linear energy transfer (high-LET) ionizing radiation (IR) is a promising cancer treatment modality owing to advantageous physical properties of energy deposition and associated toxicity over X-rays. Therapies utilizing high-LET radiation will benefit from a better understanding of the molecular mechanisms underpinning their increased biological efficacy. Towards this goal, we investigate here the biological consequences of well-defined clusters of DNA double-strand breaks (DSBs), a form of DNA damage, which on theoretical counts, has often been considered central to the enhanced toxicity of high-LET IR. We test clonal cell lines harboring in their genomes constructs with appropriately engineered I-SceI recognition sites that convert upon I-SceI expression to individual DSBs, or DSB-clusters comprising known numbers of DSBs with defined DNA-ends. We find that, similarly to high-LET IR, DSB-clusters of increasing complexity, i.e. increasing numbers of DSBs, with compatible or incompatible ends, compromise classical non-homologous end-joining, favor DNA end-resection and promote resection-dependent DSB-processing. Analysis of RAD51 foci shows increased engagement of error-free homologous recombination on DSB-clusters. Multicolor fluorescence in situ hybridization analysis shows that complex DSB-clusters markedly increase the incidence of structural chromosomal abnormalities (SCAs). Since RAD51-knockdown further increases SCAs-incidence, we conclude that homologous recombination suppresses SCAs-formation. Strikingly, CtIP-depletion inhibits SCAs-formation, suggesting that it relies on alternative end-joining or single-strand annealing. Indeed, ablation of RAD52 causes a marked reduction in SCAs, as does also inhibition of PARP1. We conclude that increased DSB-cluster formation that accompanies LET-increases, enhances IR-effectiveness by promoting DNA end-resection, which suppresses c-NHEJ and enhances utilization of alt-EJ or SSA. Although increased resection also favors HR, on balance, error-prone processing dominates, causing the generally observed increased toxicity of high-LET radiation. These findings offer new mechanistic insights into high-LET IR-toxicity and have translational potential in the clinical setting that may be harnessed by combining high-LET IR with inhibitors of PARP1 or RAD52.

DNA-PKcs kinase activity orchestrates both end-processing and end-ligation.

Recent structural studies have revealed the atomic details of how the DNA-PKcs kinase protects DNA ends, recruits and activates Artemis endonuclease for end-processing, and phosphorylates itself for end-ligation, revealing the molecular details from end-processing to end-ligation, two critical phases of the non-homologous end-joining (NHEJ) DNA-double strand break repair pathway.

Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase.

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3'-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3'-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP -/- HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3'-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3'-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3'-phosphate-terminated and fewer 3'-hydroxyl-terminated DSBs than parental cells 5-15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3'-phosphatase, loss of PNKP significantly sensitizes cells to 3'-phosphate-terminated DSBs, due to a 3'-dephosphorylation defect.