Cold snapshots of DNA repair: Cryo-EM structures of DNA-PKcs and NHEJ machinery.

The proteins and protein assemblies involved in DNA repair have been the focus of a multitude of structural studies for the past few decades. Historically, the structures of these protein complexes have been resolved by X-ray crystallography. However, more recently with the advancements in cryo-electron microscopy (cryo-EM) ranging from optimising the methodology for sample preparation to the development of improved electron detectors, the focus has shifted from X-ray crystallography to cryo-EM. This methodological transition has allowed for the structural determination of larger, more complex protein assemblies involved in DNA repair pathways and has subsequently led to a deeper understanding of the mechanisms utilised by these fascinating molecular machines. Here, we review some of the key structural advancements that have been gained in the study of non-homologous end joining (NHEJ) by the use of cryo-EM, with a focus on assemblies composed of DNA-PKcs and Ku70/80 (Ku) and the various methodologies utilised to obtain these structures.

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

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.

Surface Passivation with a Perfluoroalkane Brush Improves the Precision of Single-Molecule Measurements.

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous-aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces.

Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5'-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

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.

COSMIC Cancer Gene Census 3D database: understanding the impacts of mutations on cancer targets.

Mutations in hallmark genes are believed to be the main drivers of cancer progression. These mutations are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC). Structural appreciation of where these mutations appear, in protein-protein interfaces, active sites or deoxyribonucleic acid (DNA) interfaces, and predicting the impacts of these mutations using a variety of computational tools are crucial for successful drug discovery and development. Currently, there are 723 genes presented in the COSMIC Cancer Gene Census. Due to the complexity of the gene products, structures of only 87 genes have been solved experimentally with structural coverage between 90% and 100%. Here, we present a comprehensive, user-friendly, web interface (https://cancer-3d.com/) of 714 modelled cancer-related genes, including homo-oligomers, hetero-oligomers, transmembrane proteins and complexes with DNA, ribonucleic acid, ligands and co-factors. Using SDM and mCSM software, we have predicted the impacts of reported mutations on protein stability, protein-protein interfaces affinity and protein-nucleic acid complexes affinity. Furthermore, we also predicted intrinsically disordered regions using DISOPRED3.

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.

Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis.

Resolution advances in cryoelectron microscopy (cryo-EM) now offer the possibility to visualize structural effects of naturally occurring resistance mutations in proteins and also of understanding the binding mechanisms of small drug molecules. In Mycobacterium tuberculosis the multifunctional heme enzyme KatG is indispensable for activation of isoniazid (INH), a first-line pro-drug for treatment of tuberculosis. We present a cryo-EM methodology for structural and functional characterization of KatG and INH resistance variants. The cryo-EM structure of the 161 kDa KatG dimer in the presence of INH is reported to 2.7 Å resolution allowing the observation of potential INH binding sites. In addition, cryo-EM structures of two INH resistance variants, identified from clinical isolates, W107R and T275P, are reported. In combination with electronic absorbance spectroscopy our cryo-EM approach reveals how these resistance variants cause disorder in the heme environment preventing heme uptake and retention, providing insight into INH resistance.

Dimers of DNA-PK create a stage for DNA double-strand break repair.

DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.

Stages, scaffolds and strings in the spatial organisation of non-homologous end joining: Insights from X-ray diffraction and Cryo-EM.

Non-homologous end joining (NHEJ) is the preferred pathway for the repair of DNA double-strand breaks in humans. Here we describe three structural aspects of the repair pathway: stages, scaffolds and strings. We discuss the orchestration of DNA repair to guarantee robust and efficient NHEJ. We focus on structural studies over the past two decades, not only using X-ray diffraction, but also increasingly exploiting cryo-EM to investigate the macromolecular assemblies.

Genomics, Computational Biology and Drug Discovery for Mycobacterial Infections: Fighting the Emergence of Resistance.

Tuberculosis (TB) and leprosy are mycobacterial infections caused by Mycobacterium tuberculosis and Mycobacterium leprae respectively. These diseases continue to be endemic in developing countries where the cost of new medicines presents major challenges. The situation is further exacerbated by the emergence of resistance to many front-line antibiotics. A priority now is to design new antimycobacterials that are not only effective in combatting the diseases but are also less likely to give rise to resistance. In both these respects understanding the structure of drug targets in M. tuberculosis and M. leprae is crucial. In this review we describe structure-guided approaches to understanding the impacts of mutations that give rise to antimycobacterial resistance and the use of this information in the design of new medicines.

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV =O Formation.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

Druggable binding sites in the multicomponent assemblies that characterise DNA double-strand-break repair through non-homologous end joining.

Non-homologous end joining (NHEJ) is one of the two principal damage repair pathways for DNA double-strand breaks in cells. In this review, we give a brief overview of the system including a discussion of the effects of deregulation of NHEJ components in carcinogenesis and resistance to cancer therapy. We then discuss the relevance of targeting NHEJ components pharmacologically as a potential cancer therapy and review previous approaches to orthosteric regulation of NHEJ factors. Given the limited success of previous investigations to develop inhibitors against individual components, we give a brief discussion of the recent advances in computational and structural biology that allow us to explore different targets, with a particular focus on modulating protein-protein interaction interfaces. We illustrate this discussion with three examples showcasing some current approaches to developing protein-protein interaction inhibitors to modulate the assembly of NHEJ multiprotein complexes in space and time.

A subtle structural change in the distal haem pocket has a remarkable effect on tuning hydrogen peroxide reactivity in dye decolourising peroxidases from Streptomyces lividans.

Dye decolourising peroxidases (DyPs) are oxidative haem containing enzymes that can oxidise organic substrates by first reacting with hydrogen peroxide. Herein, we have focused on two DyP homologs, DtpAa and DtpA, from the soil-dwelling bacterium Streptomyces lividans. By using X-ray crystallography, stopped-flow kinetics, deuterium kinetic isotope studies and EPR spectroscopy, we show that both DyPs react with peroxide to form compound I (a FeIV[double bond, length as m-dash]O species and a porphyrin π-cation radical), via a common mechanism, but the reactivity and rate limits that define the mechanism are markedly different between the two homologs (DtpA forms compound I rapidly, no kinetic isotope effect; DtpAa 100-fold slower compound I formation and a distinct kinetic isotope effect). By determining the validated ferric X-ray structure of DtpAa and comparing it with the ferric DtpA structure, we attribute the kinetic differences to a subtle structural repositioning of the distal haem pocket Asp side chain. Through site-directed mutagenesis we show the acid-base catalyst responsible for proton-transfer to form compound I comprises a combination of a water molecule and the distal Asp. Compound I formation in the wild-type enzymes as well as their distal Asp variants is pH dependent, sharing a common ionisation equilibrium with an apparent pKa of ∼4.5-5.0. We attribute this pKa to the deprotonation/protonation of the haem bound H2O2. Our studies therefore reveal a mechanism for compound I formation in which the rate limit may be shifted from peroxide binding to proton-transfer controlled by the distal Asp position and the associated hydrogen-bonded water molecules.

Structural biology of multicomponent assemblies in DNA double-strand-break repair through non-homologous end joining.

The mechanisms mediating the repair of DNA damage in human cells have been the focus of a multitude of studies since the middle of the previous century, and many of the proteins implicated in these processes have been identified as being part of large macromolecular assemblies. This review gives an overview of the current knowledge of protein structures specifically involved in the repair of DNA double strand breaks through Non-Homologous End Joining, with a focus on recent structures obtained via cryo-electron microscopy and prospects for how this rapidly evolving method will impact our understanding of DNA repair.

High-throughput structures of protein-ligand complexes at room temperature using serial femtosecond crystallography.

High-throughput X-ray crystal structures of protein-ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein-ligand complexes using SFX.