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

Cryo-EM as a Tool to Study the Diverse Interactome of Ku 70/80 in NHEJ and Beyond

This PhD thesis focuses on Non-Homologous End Joining (NHEJ), one of the main DNA repair mechanisms in cells. NHEJ lies under the umbrella of the DNA Damage Response (DDR), a collection of cellular pathways that combat the tens of thousands DNA lesions a single cell experiences each day from both endogenous and exogenous sources (1). NHEJ, alongside other DNA repair pathways, is responsible for the repair of a subtype of DNA damage, defined as double-strand breaks (DSBs) (2, 3). Although less frequent than other types of damage, DSBs are the most perilous (3). If remained unrepaired or repaired incorrectly, they can be drivers for chromosomal rearrangements, cellular senescence, and apoptosis (2, 4). Despite its fundamental role in genomic stability, NHEJ has been implicated in carcinogenesis, tumour progression and radiotherapy and chemotherapy resistance (5). Since the 1980s when NHEJ was discovered, biochemical, cellular, and structural studies have led to a greater understanding of how the pathway progresses and the nature of the components involved. Briefly, DSBs are initially recognised by the Ku 70/80 (Ku) heterodimer (6), which subsequently recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme complex, to mediate synapsis of the broken DNA ends (7, 8). Depending on the DNA end configuration, DNA end processing can follow by specialised processing enzymes, nucleases, and DNA polymerases to ensure ends are ready for ligation (9). Ligation is mediated by the DNA Ligase IV and X-ray cross-complementing protein 4 (XRCC4) complex (10), with the help of the XRCC4-like factor (XLF), which forms stabilising filaments with XRCC4 (11). Additional accessory factors also display scaffolding or enzymatic roles. However, the more we dive into NHEJ, the more we understand how highly complex this process is. NHEJ is very dynamic, and over the years, various scenarios of its assembly have been proposed. The proteins’ exact arrangement during pathway progression remains still largely unclear. Therefore, the aim of my work lies in further understanding the interactions that take place for efficient DSB repair by NHEJ. To achieve this, I have focused on further elucidating the interactome of the DSB recognition component, Ku. Ku bound to DSBs is regarded as the interaction hub of NHEJ (12-14), responsible for the recruitment of multiple NHEJ components to the site of damage. Deciphering the nature of these interactions should reveal how these components are arranged in space and time to mediate DSB repair but could also prove a fruitful avenue for specifically targeting NHEJ in a cancer setting. Despite strong biochemical and cellular evidence of these interactions, there is limited information on their structural arrangement. Benefiting from the “resolution revolution” of cryo-electron microscopy (cryo-EM) (15), during my PhD work, I aimed at elucidating the structural basis of the interactions of Ku with DNA and different NHEJ components. Interestingly, in the first cryo-EM models of Ku alone and bound to DNA and subsequent NHEJ complexes containing Ku, I was able to visualise the detailed molecular basis of its interaction with a previously identified but not fully examined small molecule stimulator of NHEJ, inositol hexakisphosphate (IP6) (16, 17), which co-purified alongside Ku. Following that, in collaboration with members of our group and cryo-EM facilities in Cambridge, the diverse interactome of Ku with NHEJ components such as DNA-PK, the Ligase IV-XRCC4 complex and XLF was visualised in the context of different multi-component assemblies, defined as NHEJ supercomplexes. Further attempts to obtain cryo-EM models of Ku with additional non-core NHEJ components were carried out, with some allowing the visualisation of high-resolution structures, such as that of Ku with the accessory factor Paralog of XRCC4 and XLF (PAXX) and other revealing the dimeric arrangement of NHEJ processing enzymes as DNA polymerase λ. Through collaborations with external academic and industrial partners, the importance of the identified interactions was further confirmed using biochemical, biophysical, and cellular studies. Beyond the NHEJ machinery, I aimed to examine the interactions of Ku with nucleosomes of different DNA lengths in an effort to move away from “naked” DNA and understand how NHEJ would assemble in a chromatin environment. Finally, given the implications of NHEJ in resistance to radiotherapy and chemotherapy, I aimed to develop a drug discovery pipeline to examine the druggability of protein-protein interactions (PPIs) in NHEJ, given their diversity and crucial role in pathway progression, as seen from our cryo-EM work. I focused on one interaction, that of Ku with XLF. Through in silico docking and subsequent experimental validation, I was able to identify two promising hits which could act as a basis for the development of inhibitors. In summary, the work presented in this PhD thesis describes the power provided by cryo-EM in allowing us to further elucidate the molecular basis of Ku’s interactome and its importance in pathway progression and a new avenue in exploring NHEJ PPIs as potential drug target

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

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.Wellcome Trust for a Programme Grant (O93167/Z/10/Z; 2011–2016) and Investigator Award (200814/Z/16/Z; 2016 -

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.Wellcome Trust for a Programme Grant (O93167/Z/10/Z; 2011–2016) and Investigator Award (200814/Z/16/Z

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

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

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

International audienceThe 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

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

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The 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

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

Acknowledgements: JBC thank the I2BC platforms for Interactions of Macromolecules (PIM) and for Crystallization supported by French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INBS-0005. We thank the HTX platform at EMBL, Grenoble. We thank Magali Aumont-Nicaise and Adrian Velazquez-Campoy for their help with ITC data. AG was supported by a CIFRE (ANRT) fellowship. JBC was supported by ANR-20-CE11-0026, ANR-17-CE12-0020 and ANR-21-CE12-0019-01. We also thank the staff from PROXIMA-1, PROXIMA-2a and SWING beamlines for their help in data collection and synchrotron SOLEIL (Saint-Aubin, France) for provision of synchrotron radiation facilities. SJ, MJM, and ER thank Mauro Modesti for providing SNAP tag Ku protein. PC, SB and PF were supported by the Ligue Contre le Cancer as Equipe Labellisée 2018 and ANR-20-CE11-0026. PC is a scientist from INSERM. We acknowledge the TRI imaging facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04). We would like to thank the Imaging Core Facility TRI-IPBS, in particular Antonio Peixoto and Eve Pitot for technical assistance. A.C., A.K. and T.L.B. thank the Wellcome Trust for support of this research through the award of an Investigator Award (200814/Z/16/Z; 2016–2022).Funder: Ligue Contre le Cancer; DOI: https://doi.org/10.13039/501100004099The 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