Structure of an activated DNA-PK and its implications for NHEJ.

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 Å overall and 3.2 Å for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.

Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.

Classical non-homologous end joining (cNHEJ) and homologous recombination compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/ß domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

Discovery and development of novel DNA-PK inhibitors by targeting the unique Ku-DNA interaction.

DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) repair pathway and the DNA damage response (DDR). DNA-PK has therefore been pursued for the development of anti-cancer therapeutics in combination with ionizing radiation (IR). We report the discovery of a new class of DNA-PK inhibitors that act via a novel mechanism of action, inhibition of the Ku-DNA interaction. We have developed a series of highly potent and specific Ku-DNA binding inhibitors (Ku-DBi's) that block the Ku-DNA interaction and inhibit DNA-PK kinase activity. Ku-DBi's directly interact with the Ku and inhibit in vitro NHEJ, cellular NHEJ, and potentiate the cellular activity of radiomimetic agents and IR. Analysis of Ku-null cells demonstrates that Ku-DBi's cellular activity is a direct result of Ku inhibition, as Ku-null cells are insensitive to Ku-DBi's. The utility of Ku-DBi's was also revealed in a CRISPR gene-editing model where we demonstrate that the efficiency of gene insertion events was increased in cells pre-treated with Ku-DBi's, consistent with inhibition of NHEJ and activation of homologous recombination to facilitate gene insertion. These data demonstrate the discovery and application of new series of compounds that modulate DNA repair pathways via a unique mechanism of action.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

The Multifaceted Roles of Ku70/80.

DNA double-strand breaks (DSBs) are accidental lesions generated by various endogenous or exogenous stresses. DSBs are also genetically programmed events during the V(D)J recombination process, meiosis, or other genome rearrangements, and they are intentionally generated to kill cancer during chemo- and radiotherapy. Most DSBs are processed in mammalian cells by the classical nonhomologous end-joining (c-NHEJ) pathway. Understanding the molecular basis of c-NHEJ has major outcomes in several fields, including radiobiology, cancer therapy, immune disease, and genome editing. The heterodimer Ku70/80 (Ku) is a central actor of the c-NHEJ as it rapidly recognizes broken DNA ends in the cell and protects them from nuclease activity. It subsequently recruits many c-NHEJ effectors, including nucleases, polymerases, and the DNA ligase 4 complex. Beyond its DNA repair function, Ku is also involved in several other DNA metabolism processes. Here, we review the structural and functional data on the DNA and RNA recognition properties of Ku implicated in DNA repair and in telomeres maintenance.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors.

DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase.

Cryo-EM structure of the DNA-PK holoenzyme.

DNA-dependent protein kinase (DNA-PK) is a large protein complex central to the nonhomologous end joining (NHEJ) DNA-repair pathway. It comprises the DNA-PK catalytic subunit (DNA-PKcs) and the heterodimer of DNA-binding proteins Ku70 and Ku80. Here, we report the cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs at 4.4-Å resolution and the DNA-PK holoenzyme at 5.8-Å resolution. The DNA-PKcs structure contains three distinct segments: the N-terminal region with an arm and a bridge, the circular cradle, and the head that includes the kinase domain. Two perpendicular apertures exist in the structure, which are sufficiently large for the passage of dsDNA. The DNA-PK holoenzyme cryo-EM map reveals density for the C-terminal globular domain of Ku80 that interacts with the arm of DNA-PKcs. The Ku80-binding site is adjacent to the previously identified density for the DNA-binding region of the Ku70/Ku80 complex, suggesting concerted DNA interaction by DNA-PKcs and the Ku complex.

Mapping the Ku Interactome Using Proximity-Dependent Biotin Identification in Human Cells.

The Ku heterodimer, composed of Ku70 and Ku80, is best characterized for its role in repairing double-stranded DNA breaks but is also known to participate in other regulatory processes. Despite our understanding of Ku protein interplay during DNA repair, the extent of Ku's protein interactions in other processes has never been fully determined. Using proximity-dependent biotin identification (BioID) and affinity purification coupled to mass spectrometry (AP-MS) with wild-type Ku70, we identified candidate proteins that interact with the Ku heterodimer in HEK293 cells, in the absence of exogenously induced DNA damage. BioID analysis identified approximately 250 nuclear proteins, appearing in at least two replicates, including known Ku-interacting factors such as MRE11A, WRN, and NCOA6. Meanwhile, AP-MS analysis identified approximately 50 candidate proteins. Of the novel protein interactors identified, many were involved in functions already suspected to involve Ku such as transcriptional regulation, DNA replication, and DNA repair, while several others suggest that Ku may be involved in additional functions such as RNA metabolism, chromatin-remodeling, and microtubule dynamics. Using a combination of BioID and AP-MS, this is the first report that comprehensively characterizes the Ku protein interaction landscape, revealing new cellular processes and protein complexes involving the Ku complex.

Ku70 N-terminal lysines acetylation/deacetylation is required for radiation-induced DNA-double strand breaks repair.

Ku70 protein in hetero-trimeric complex with Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) represents a critical component of the nonhomologous-end-joining (NHEJ), the major machinery of DSBs repair in mammalian cells. It has been previously shown that modulation of Ku70 acetylation by histone deacetylases (HDAC) inhibitors induced sensitization of cancer cells to chemotherapeutic agents. However, the effects of such modulation on the repair of Ionizing Radiation (IR)-induced DSBs and the importance of dynamic equilibrium of acetylation/deacetylation have not been studied in details. To address these questions aceto-blocking and aceto-mimicking mutants were designed by replacing Ku70 lysine residues K317, K331 and K338 with arginine and glutamine respectively via site-directed mutagenesis. Transformed human embryonic lung fibroblasts MRC5VA were transfected to create stables cells lines over-expressing Ku70 mutant proteins. Clonogenic survival and γ-H2AX foci assays were performed to study the impact of these mutants on DNA repair proficiency of MRC5VA cells in response to IR. We report here that both Ku70 aceto-blocking and aceto-mimicking mutants rendered MRC5VA cells more susceptible to IR in terms of clonogenic survival and γH2AX foci. Moreover, modelling the possible interactions and structural impact of these aceto-blocking and aceto-mimicking mutants with DNA substrate showed that mimicking acetylation/deacetylation of K331 and K338 could directly compromise KU-DNA interactions, whereas K317 may have a more subtle role via forming a salt bridge with E330 thus optimising the positioning of the helix containing both K331 and K338 residues on the DNA. Our data indicates that dynamic equilibrium of acetylation/deacetylation of Ku70 lysine residues K317, K331 and K338 is critical for optimal repair of IR-induced DSBs, and may offer a novel therapeutic approach for cancer treatment.

Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency.

The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase µ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli.

Mycobacterium tuberculosis and Mycobacterium smegmatis express a Ku protein and a DNA ligase D and are able to repair DNA double strand breaks (DSBs) by non-homologous end-joining (NHEJ). This pathway protects against DNA damage when bacteria are in stationary phase. Mycobacterium marinum is a member of this mycobacterium family and like M. tuberculosis is pathogenic. M. marinum lives in water, forms biofilms and infects fish and frogs. M. marinum is a biosafety level 2 (BSL2) organism as it can infect humans, although infections are limited to the skin. M. marinum is accepted as a model to study mycobacterial pathogenesis, as M. marinum and M. tuberculosis are genetically closely related and have similar mechanisms of survival and persistence inside macrophage. The aim of this study was to determine whether M. marinum could be used as a model to understand M. tuberculosis NHEJ repair. We identified and cloned the M. marinum genes encoding NHEJ proteins and generated E. coli strains that express the M. marinum Ku (Mm-Ku) and ligase D (Mm-Lig) individually or together (LHmKumLig strain) from expression vectors integrated at phage attachment sites in the genome. We demonstrated that Mm-Ku and Mm-Lig are both required to re-circularize Cla I-linearized plasmid DNA in E. coli. We compared repair of strain LHmKumLig with that of an E. coli strain (BWKuLig#2) expressing the M. tuberculosis Ku (Mt-Ku) and ligase D (Mt-Lig), and found that LHmKumLig performed 3.5 times more repair and repair was more accurate than BWKuLig#2. By expressing the Mm-Ku with the Mt-Lig, or the Mt-Ku with the Mm-Lig in E. coli, we have shown that the NHEJ proteins from M. marinum and M. tuberculosis can function together to join DNA DSBs. NHEJ repair is therefore conserved between the two species. Consequently, M. marinum is a good model to study NHEJ repair during mycobacterial pathogenesis.

A nanomedicine based combination therapy based on QLPVM peptide functionalized liposomal tamoxifen and doxorubicin against Luminal A breast cancer.

Though combination chemotherapy or antitumor nanomedicine is extensively investigated, their combining remains in infancy. Additionally, enhanced delivery of estrogen or its analogs to tumor with highly-expressed estrogen-receptor (ER) is seldom considered, despite its necessity for ER-positive breast cancer treatment. Here, nanomedicine based combination therapy using QLPVM conjugated liposomal tamoxifen (TAM) and doxorubicin (DOX) was designed and testified, where the penta-peptide was derived from Ku70 Bax-binding domain. Quantitative, semi-quantitative and qualitative approaches demonstrated the enhanced endocytosis and cytotoxicity of QLPVM conjugated sterically stabilized liposomes (QLPVM-SSLs) in vitro and in vivo. Mechanism studies of QLPVM excluded the possible electrostatic, hydrophobic or receptor-ligand interactions. However, as a weak cell-penetrating peptide, QLPVM significantly induced drug release from QLPVM-SSLs during their interaction with cells, which was favorable for drug internalization. These findings suggested that the nanomedicine based combination therapy using QLPVM-SSL-TAM and QLPVM-SSL-DOX might provide a rational strategy for Luminal A breast cancer. FROM THE CLINICAL EDITOR: Breast cancer remains a leading cause of mortality in women worldwide. Although combined therapy using hormonal antagonist and chemotherapy is the norm nowadays, the use of these agents together in a single delivery system has not been tested. Here, the authors investigated this approach using QLPVM conjugated liposomes in in-vitro and in-vivo models. The positive findings may provide a novel direction for breast cancer treatment in the near future.

In silico and in vitro characterization of the mycobacterial protein Ku to unravel its role in non-homologous end-joining DNA repair.

Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.

Measuring protein stoichiometry with single-molecule imaging in Xenopus egg extracts.

In human cells, DNA double-strand breaks are rapidly bound by the highly abundant non-homologous end joining (NHEJ) factor Ku70/Ku80 (Ku). Cellular imaging and structural data revealed a single Ku molecule is bound to a free DNA end and yet the mechanism regulating Ku remains unclear. Here, we describe how to utilize the cell-free Xenopus laevis egg extract system in conjunction with single-molecule microscopy to investigate regulation of Ku stoichiometry during non-homologous end joining. Egg extract is an excellent model system to study DNA repair as it contains the soluble proteome including core and accessory NHEJ factors, and efficiently repairs double-strand breaks in an NHEJ-dependent manner. To examine the Ku stoichiometry in the extract system, we developed a single-molecule photobleaching assay, which reports on the number of stable associated Ku molecules by monitoring the intensity of fluorescently labeled Ku molecules bound to double-stranded DNA over time. Photobleaching is distinguishable as step decreases in fluorescence intensity and the number of photobleaching events indicate fluorophore stoichiometry. In this paper we describe sample preparation, experimental methodology, and data analysis to discern Ku stoichiometry and the regulatory mechanism controlling its loading. These approaches can be readily adopted to determine stoichiometry of molecular factors within other macromolecular complexes.

SAP domain forms a flexible part of DNA aperture in Ku70/80.

Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-bound state. The second position, which was observed in both apo and DNA-bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. DATABASES: EM maps have been deposited in EMDB (EMD-11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.

X-ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double-strand break repair impacting cell and cancer biology.

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIVBRCT -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.

Ligand binding characteristics of the Ku80 von Willebrand domain.

The N-terminal von Willebrand domain of Ku80 supports interactions with a Ku binding motif (KBM) that has been identified in at least three other DNA repair proteins: the non-homologous end joining (NHEJ) scaffold APLF, the modulator of retrovirus infection, MRI, and the Werner syndrome protein (WRN). A second, more recently identified Ku binding motif present in XLF and several other proteins (KBMX) has also been reported to interact with this domain. The isolated Ku80 von Willebrand antigen domain (vWA) from Xenopus laevis has a sequence that is 60% identical with the human domain, is readily expressed and has been used to investigate these interactions. Structural characterization of the complexes formed with the KBM motifs in human APLF, MRI, and WRN identify a conserved binding site that is consistent with previously-reported mutational studies. In contrast with the KBM binding site, structural studies indicate that the KBMX site is occluded by a distorted helix. Fluorescence polarization and 19F NMR studies of a fluorinated XLF C-terminal peptide failed to indicate any interaction with the frog vWA. It was hypothesized that availability of this binding site is conditional, i.e., dependent on specific experimental conditions or other repair factors to make the site available for binding. Modulating the fraction of KBMX-accessible binding site mutationally demonstrated that the more open site is capable of binding the KBMXXLF motif peptide. It is suggested that the conditional nature of KBMX binding limits formation of non-productive complexes so that activation-dependent site availability can more optimally support advancing the synapsis process.