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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.

DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3′-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg 2+ -reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg 2+ , and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage. DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. Here the authors present X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA that unveil determinants of LIG1 substrate recognition and enzymatic fidelity.

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5′ overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9–mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11’s nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

The telomere end-protection problem is defined by the aggregate of DNA damage signaling and repair pathways that require repression at telomeres. To define the end-protection problem, we removed the whole shelterin complex from mouse telomeres through conditional deletion of TRF1 and TRF2 in nonhomologous end-joining (NHEJ) deficient cells. The data reveal two DNA damage response pathways not previously observed upon deletion of individual shelterin proteins. The shelterin-free telomeres are processed by microhomology-mediated a Iter nati ve-NHEJ when Ku70/80 is absent and are attacked by nucleolytic degradation in the absence of 53BP1. The data establish that the end-protection problem is specified by six pathways [ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) signaling, classical-NHEJ, alt-NHEJ, homologous recombination, and resection] and show how shelterin acts with general DNA damage response factors to solve this problem.

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

Mitochondrial dysfunction has been linked to myriad human diseases and toxicant exposures, highlighting the need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide and oligomycin (ATP-synthase inhibitors), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we measured the fundamental parameters of mitochondrial respiratory chain function: basal oxygen consumption, ATP-linked respiration, maximal respiratory capacity, spare respiratory capacity and proton leak in the model organism Caenhorhabditis elegans. Since mutations in mitochondrial homeostasis genes cause mitochondrial dysfunction and have been linked to human disease, we measured mitochondrial respiratory function in mitochondrial fission (drp-1)-, fusion (fzo-1)-, mitophagy (pdr-1, pink-1)-, and electron transport chain complex III (isp-1)-deficient C. elegans. All showed altered function, but the nature of the alterations varied between the tested strains. We report increased basal oxygen consumption in drp-1; reduced maximal respiration in drp-1, fzo-1, and isp-1; reduced spare respiratory capacity in drp-1 and fzo-1; reduced proton leak in fzo-1 and isp-1; and increased proton leak in pink-1 nematodes. As mitochondrial morphology can play a role in mitochondrial energetics, we also quantified the mitochondrial aspect ratio for each mutant strain using a novel method, and for the first time report increased aspect ratios in pdr-1- and pink-1-deficient nematodes.

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4¹⁻¹⁵⁷-Cernunnos¹⁻²²⁴ complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4¹⁻¹⁵⁷ and Cernunnos¹⁻²²⁴ homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu⁵⁵, Asp⁵⁸, Met⁶¹, and Phe¹⁰⁶) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.

The MYC family of cellular oncogenes includes c-Myc, N-myc, and L-myc, which encode transcriptional regulators involved in the control of cell proliferation and death. Accordingly, these genes become aberrantly activated and expressed in specific types of cancers. For example, c-Myc translocations occur frequently in human B lymphoid tumors, while N-myc gene amplification is frequent in human neuroblastomas. The observed association between aberrations in particular MYC family genes and specific subsets of malignancies might reflect, at least in part, tissue-specific differences in expression or function of a given MYC gene. Since c-Myc and N-myc share substantial functional redundancy, another factor that could influence tumor-specific gene activation would be mechanisms that target aberrations (e.g., translocations) in a given MYC gene in a particular tumor progenitor cell type. We have previously shown that mice deficient for the DNA Ligase4 (Lig4) nonhomologous DNA end-joining factor and the p53 tumor suppressor routinely develop progenitor (pro)-B cell lymphomas that harbor translocations leading to c-Myc amplification. Here, we report that a modified allele in which the c-Myc coding sequence is replaced by N-myc coding sequence (NCR allele) competes well with the wild-type c-Myc allele as a target for oncogenic translocations and amplifications in the Lig4/p53-deficient pro-B cell lymphoma model. Tumor onset, type, and cytological aberrations are similar in tumors harboring either the wild-type c-Myc gene or the NCR allele. Our results support the notion that particular features of the c-Myc locus select it as a preferential translocation/amplification target, compared to the endogenous N-myc locus, in Lig4/p53-deficient pro-B cell lymphomas.