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DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy., (© 2024. The Author(s).)

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis - trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

The three human SNM1 metallo-β-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated., (© 2024. The Author(s).)

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers., Competing Interests: B. L. P. is a co-founder and/or member of the scientific advisory board of several companies focusing on the development of protein and peptide therapeutics. J. C. K. W., A. M., H. T. B., L. J. G. C., F. E. M., I. F., D. L. E., Q. H. and A. H. N. are employees of Calico Life Sciences LLC. A provisional patent disclosure was filed regarding the methodology and compounds described in this study., (This journal is © The Royal Society of Chemistry.)

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5'-to-3' exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the 'unhooking' step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Nine modified nucleosides, incorporating zinc-binding pharmacophores, have been synthesised and evaluated as inhibitors of the DNA repair nuclease SNM1A. The series included oxyamides, hydroxamic acids, hydroxamates, a hydrazide, a squarate ester and a squaramide. A hydroxamic acid-derived nucleoside inhibited the enzyme, offering a novel approach for potential therapeutic development through the use of rationally designed nucleoside derived inhibitors.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.