Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.

Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ.

The ability of humans to maintain the integrity of the genome is imperative for cellular survival. DNA double-strand breaks (DSBs) are considered the most critical type of DNA lesion, which can ultimately lead to diseases including cancer. Non-homologous end joining (NHEJ) is one of two core mechanisms utilized to repair DSBs. DNA-PK is a key component in this process and has recently been shown to form alternate long-range synaptic dimers. This has led to the proposal that these complexes can be formed before transitioning to a short-range synaptic complex. Here we present cryo-EM data representing an NHEJ supercomplex consisting of a trimer of DNA-PK in complex with XLF, XRCC4, and DNA Ligase IV. This trimer represents a complex of both long-range synaptic dimers. We discuss the potential role of the trimeric structure, and possible higher order oligomers, as structural intermediates in the NHEJ mechanism, or as functional DNA repair centers.

PAXX binding to the NHEJ machinery explains functional redundancy with XLF.

Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells.

Two distinct long-range synaptic complexes promote different aspects of end processing prior to repair of DNA breaks by non-homologous end joining.

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.

Unravelling the complexities of DNA-PK activation by structure-based mutagenesis.

It has been known for decades that the DNA-dependent protein kinase (DNA-PK) is only an active serine/threonine protein kinase when it is bound to a DNA double-stranded end; still, the molecular details of how this activation is achieved have remained elusive. The recent surge in structural information for DNA-PK complexes has provided valuable insights into the process of DNA end recognition by DNA-PK. A particularly intriguing feature of this kinase is a region of the protein that can transition from a seemingly structurally disordered state to a single alpha-helix that traverses down the DNA binding cradle. The DNA-PK bound DNA end of the DNA substrate engages with and appears to split around this helix which has been named the DNA End Blocking helix (DEB). Here a mutational approach is utilized to clarify the role of the DEB, and how DNA ends activate the enzyme. Our data suggest two distinct methods of kinase activation that is dependent on the DNA end chemistry. If the DNA end can split around the helix and stabilize the interaction between the DNA end and the DEB with a recently defined Helix-Hairpin-Helix (HHH) motif, the kinase forms an end-protection monomer that is active towards DNA-PK's many substrates. But if the DNA end cannot stably interact with the DEB [because of the DNA end structure, for instance hairpins, or because the DEB has been disrupted by mutation], the kinase is only partially activated, resulting in specific autophosphorylations of the DNA-PK monomer that allows nucleolytic end-processing. We posit that mutants that disrupt the capacity to stably generate the DEB/HHH DNA end-interaction are inefficient in generating the dimer complex that is requisite for NHEJ. In support of this idea, mutations that promote formation of this dimer partially rescue the severe cellular phenotypes associated with mutation of the DEB helix.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).

Identification of a Hypomorphic FANCG Variant in Bernese Mountain Dogs.

Bernese mountain dogs (BMDs), have an overall cancer incidence of 50%, half of which is comprised of an otherwise rare tumor, histiocytic sarcoma (HS). While recent studies have identified driver mutations in the MAPK pathway, identification of key predisposing genes has been elusive. Studies have identified several loci to be associated with predisposition to HS in BMDs, including near the MTAP/CDKN2A region, but no causative coding variant has been identified. Here we report the presence of a coding polymorphism in the gene encoding FANCG, near the MTAP/CDKN2A locus. This variant is in a conserved region of the protein and appears to be specific to BMDs. Canine fibroblasts derived from dogs homozygous for this variant are hypersensitive to cisplatin. We show this canine FANCG variant and a previously defined hypomorphic FANCG allele in humans impart similar defects in DNA repair. However, our data also indicate that this variant is neither necessary nor sufficient for the development of HS. Furthermore, BMDs homozygous for this FANCG allele display none of the characteristic phenotypes associated with Fanconi anemia (FA) such as anemia, short stature, infertility, or an earlier age of onset for HS. This is similar to findings in FA deficient mice, which do not develop overt FA without secondary genetic mutations that exacerbate the FA deficit. In sum, our data suggest that dogs with deficits in the FA pathway are, like mice, innately resistant to the development of FA.

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair.

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

Activation of DNA-PK by hairpinned DNA ends reveals a stepwise mechanism of kinase activation.

As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

Deciphering the role of distinct DNA-PK phosphorylations at collapsed replication forks.

It has recently been established that the marked sensitivity of ATM deficient cells to topoisomerase poisons like camptothecin (Cpt) results from unrestrained end-joining of DNA ends at collapsed replication forks that is mediated by the non-homologous end joining [NHEJ] pathway and results in the induction of copious numbers of genomic alterations, termed "toxic NHEJ". Ablation of core components of the NHEJ pathway reverses the Cpt sensitivity of ATM deficient cells, but inhibition of DNA-PKcs does not. Here, we show that complete ablation of DNA-PKcs partially reverses the Cpt sensitivity of ATM deficient cells; thus, ATM deficient cells lacking DNA-PKcs are more resistant to Cpt than cells expressing DNA-PKcs. However, the relative sensitivity of DNA-PKcs proficient ATM deficient cells is inversely proportional to DNA-PKcs expression levels. These data suggest that DNA-PK may phosphorylate an ATM target (that contributes to Cpt resistance), explaining partial rescue of Cpt sensitivity in cells expressing high levels of DNA-PKcs. Although crippling NHEJ function by mutagenic blockade of the critical ABCDE autophosphorylation sites in DNA-PKcs also sensitizes cells to Cpt, this sensitization apparently occurs by a distinct mechanism from ATM ablation because blockade of these sites actually rescues ATM deficient cells from toxic NHEJ. These data are consistent with autophosphorylation of the ABCDE sites (and not ATM mediated phosphorylation) in response to Cpt-induced damage. In contrast, blockade of S3205 (an ATM dependent phosphorylation site in DNA-PKcs) that minimally impacts NHEJ, increases Cpt sensitivity. In sum, these data suggest that ATM and DNA-PK cooperate to facilitate Cpt-induced DNA damage, and that ATM phosphorylation of S3205 facilitates appropriate repair at collapsed replication forks.

An Antiviral DNA Response without the STING?

Higher eukaryotes have evolved elegant and redundant pathways to protect their genomes from both genotoxic stressors and foreign DNA from invading pathogens. Emerging data from Burleigh et al. suggest that these distinct pathways may share factors to enhance the functional redundancy of both.

Deciphering phenotypic variance in different models of DNA-PKcs deficiency.

DNA-PKcs deficiency has been studied in numerous animal models and cell culture systems. In previous studies of kinase inactivating mutations in cell culture systems, ablation of DNA-PK's catalytic activity results in a cell phenotype that is virtually indistinguishable from that ascribed to complete loss of the enzyme. However, a recent compelling study demonstrates a remarkably more severe phenotype in mice harboring a targeted disruption of DNA-PK's ATP binding site as compared to DNA-PKcs deficient mice. Here we investigate the mechanism for these divergent results. We find that kinase inactivating DNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity. Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we present data demonstrating that DNA-PK primarily autophosphorylates.

Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells.

Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1293) is similar to the protein produced in bacteria (ALKBH1Ec) with regard to its m6A demethylase and AP lyase activities. In addition, we find that ALKBH1293 forms a covalent adduct with the 5' product of the lyase product in a manner analogous to ALKBH1Ec. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.

Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining.

XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.

PAXX Is an Accessory c-NHEJ Factor that Associates with Ku70 and Has Overlapping Functions with XLF.

In mammalian cells, classical non-homologous end joining (c-NHEJ) is critical for DNA double-strand break repair induced by ionizing radiation and during V(D)J recombination in developing B and T lymphocytes. Recently, PAXX was identified as a c-NHEJ core component. We report here that PAXX-deficient cells exhibit a cellular phenotype uncharacteristic of a deficiency in c-NHEJ core components. PAXX-deficient cells display normal sensitivity to radiomimetic drugs, are proficient in transient V(D)J recombination assays, and do not shift toward higher micro-homology usage in plasmid repair assays. Although PAXX-deficient cells lack c-NHEJ phenotypes, PAXX forms a stable ternary complex with Ku bound to DNA. Formation of this complex involves an interaction with Ku70 and requires a bare DNA extension for stability. Moreover, the relatively weak Ku-dependent stimulation of LIG4/XRCC4 activity by PAXX is unmasked by XLF ablation. Thus, PAXX plays an accessory role during c-NHEJ that is largely overlapped by XLF's function.

The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends.

The evidence that ATM affects resolution of RAG-induced DNA double-strand breaks is profuse and unequivocal; moreover, it is clear that the RAG complex itself cooperates (in an undetermined way) with ATM to facilitate repair of these double-strand breaks by the classical nonhomologous end-joining pathway. The mechanistic basis for the cooperation between ATM and the RAG complex has not been defined, although proposed models invoke ATM and RAG2's C terminus in maintaining the RAG postcleavage complex. In this study, we show that ATM reduces the rate of both coding and signal joining in a robust episomal assay; we suggest that this is the result of increased stability of the postcleavage complex. ATM's ability to inhibit VDJ joining requires its enzymatic activity. The noncore C termini of both RAG1 and RAG2 are also required for ATM's capacity to limit signal (but not coding) joining. Moreover, potential phosphorylation targets within the C terminus of RAG2 are also required for ATM's capacity to limit signal joining. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM.

Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining.

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)-deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs-deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs-deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs-deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.