Abstract Congenital Dyserythropoietic Anaemia type I (CDA-I) is a rare inherited disorder of erythropoiesis, in which erythroid cells display a unique nuclear phenotype referred to as ‘spongy’ heterochromatin. The molecular basis of CDA-I remains unknown, with most cases of CDA-I caused by mutations in CDAN1 , encoding Codanin-1, or CDIN1 , encoding for Codanin-1-interacting nuclease 1 (CDIN1). To date, very little is known about the function of CDA-I disease proteins and the mechanism by which their associated mutations cause disease. Here, we demonstrate that endogenous CDIN1 interacts with Codanin-1, to form a stable complex. Structural and functional analysis of this complex reveals that the CDIN1-Codanin-1 complex is an RNA nuclease. We shed light on the key mechanistic features of the complex using biochemical and biophysical approaches, complemented by all-atom molecular dynamics (MD) structural simulations. We identify various functional consequences of founder patient mutations on the RNA nuclease activity of CDIN1, providing a framework for understanding the pathophysiology and developing therapeutic strategies for CDA-I.

The Cockayne Syndrome B (CSB, ERCC6) protein, interacts with the exonuclease SNM1A during transcription-coupled DNA interstrand (ICL) repair, with CSB facilitating localisation of SNM1A to ICL damage. The functional and mechanistic details of this interaction in DNA repair, however, have not been defined. Here, we demonstrate that CSB enhances SNM1A resection through ICLs and identify a specific interaction between the winged-helix domain of CSB and the nuclease core of SNM1A that is crucial for recruitment and enhancement of nuclease degradation. Biochemical and single-molecule studies on DNA containing site-specific ICLs reveal that CSB increases the affinity of SNM1A to damaged DNA substrates and also alters the substrate conformation to enhance ICL processing by SNM1A. Notably, CSB was observed preferentially as a dimer when colocalised with SNM1A at ICLs, constrasting with its monomeric nature observed during repair initiation in classical transcription-coupled nucleotide excision repair. The combined results provide molecular insights into the basis of a direct contribution of CSB to a DNA repair reaction.

Impediments to faithful transcription must be resolved to ensure accurate gene expression and safeguard normal cellular function. Dedicated DNA repair pathways have therefore evolved to remove transcription-blocking DNA damage, targeted to active genes. Although significant research efforts to date have focussed on the transcription-coupled repair of bulky, UV-induced DNA damage, it is known that other forms of DNA damage can perturb RNA Polymerase II progression. Only in recent years has insight into these pathways emerged, despite the clinical significance of understanding all transcription-coupled repair pathways. These recent observations have highlighted substantial molecular differences in these pathways compared to the canonical UV-damage repair mechanisms. This review summarises our understanding to date of the molecular mechanisms that act to remove both DNA-DNA and DNA-protein crosslinks that block transcription in mammalian cells.

DNA–protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognizes its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs is still not entirely clear. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain that we named the Ubiquitin Interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains in an avidity manner. SPRTN binding to ubiquitin chains via USD leads to ∼67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. In contrast, the constitutive components of the replisome during unperturbed or translesional DNA synthesis, namely proliferating cell nuclear antigen (PCNA) or monoUb-PCNA, respectively, were poorly degraded, if at all, by SPRTN. This study reveals that the poly-ubiquitination of DPCs serves as the key signal for SPRTN’s rapid proteolysis and determines its substrate specificity towards DPCs, rather than the replisome.

The SNM1A exonuclease plays a key role in repair of interstrand crosslinks (ICLs) which represent a particularly toxic class of DNA damage. Previous work suggests that the SWI/SNF family ATP-dependent, chromatin remodeler, Cockayne Syndrome B protein (CSB) interacts with SNM1A, during transcription-coupled DNA interstrand crosslink repair (TC-ICL repair). Here, we validate this interaction using purified proteins and demonstrate that the ubiquitin-binding and winged-helix domains of CSB are required for interaction with the catalytic domain of SNM1A. The winged helix domain is essential for binding, although high-affinity SNM1A binding requires the entire CSB C-terminal region (residues 1187-1493), where two copies of the C-terminal domain of CSB are necessary for a stable interaction with SNM1A. CSB stimulates SNM1A nuclease activity on varied model DNA repair intermediate substrates. Importantly, CSB was observed to stimulate digestion through ICLs in vitro , implying a key role of the interaction in 'unhooking' during TC-ICL repair. AlphaFold3 models of CSB constructs complexed with the SNM1A catalytic domain enabled mapping of the molecular contacts required for the CSB-SNM1A interaction. This identified specific protein-protein interactions necessary for CSB's stimulation of SNM1A's activity that we confirmed experimentally. Additionally, our studies reveal the C-terminal region of CSB as a novel DNA binding region that also is involved in stimulation of SNM1A-mediated ICL repair. Moreover, targeting protein-protein interactions that are vital for specific nuclease activities, such as CSB's stimulation of SNM1A's nuclease activity, may be a productive alternative therapeutic strategy to nuclease active site inhibition.

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.

Abstract 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.

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.

In this study, the influence of a plasma electrolytic oxidation (PEO) surface treatment on a medical-grade WE43-based magnesium alloy is examined through an experimental and computational framework that considers the effects of localised corrosion features and mechanical properties throughout the corrosion process. First, a comprehensive in-vitro immersion study was performed on WE43-based tensile specimens with and without PEO surface modification, which included fully automated spatial reconstruction of the phenomenological features of corrosion through micro-CT scanning, followed by uniaxial tensile testing. Then the experimental data of both unmodified and PEO-modified groups were used to calibrate parameters of a finite element-based surface corrosion model. In-vitro, it was found that the WE43-PEO modified group had a significantly lower corrosion rate and maintained significantly higher mechanical properties than the unmodified. While corrosion rates were ∼50% lower in the WE43-PEO modified specimens, the local geometric features of corroding surfaces remained similar to the unmodified WE43 group, however evolving after almost the double amount of time. We were also able to quantitatively demonstrate that the PEO surface treatment on magnesium continued to protect samples from corrosion throughout the entire period tested, and not just in the early stages of corrosion. Using the results from the testing framework, the model parameters of the surface-based corrosion model were identified for both groups. This enabled, for the first time, in-silico prediction of the physical features of corrosion and the mechanical performance of both unmodified and PEO modified magnesium specimens. This simulation framework can enable future in-silico design and optimisation of bioabsorbable magnesium devices for load-bearing medical applications.

Formaldehyde is a pollutant and human metabolite that is toxic at high concentrations. Biological studies on formaldehyde are hindered by its high reactivity and volatility, which make it challenging to deliver quantitatively to cells. Here, we describe the development and validation of a set of N-acyloxymethylphthalimides as cell-relevant formaldehyde delivery agents. These esterase-sensitive compounds were similarly or less inhibitory to human cancer cell growth than free formaldehyde but the lead compound increased intracellular formaldehyde concentrations, increased cellular levels of thymidine derivatives (implying increased formaldehyde-mediated carbon metabolism), induced formation of cellular DNA-protein cross-links and induced cell death in pancreatic cancer cells. Overall, our N-acyloxymethylphthalimides and control compounds provide an accessible and broadly applicable chemical toolkit for formaldehyde biological research and have potential as cancer therapeutics.

This paper presents a methodology to establish a process structure property (PSP) relationship for the additive manufacturing (AM) of thin AISI 316L struts, as might be used in coronary stent applications. The methodology is based on a new geometrically based process-structure method for AM process variables, and crystal plasticity finite element (CPFE) modelling that includes the representation of melt pool microstructure morphology and texture. The effects of AM process variables are characterised with respect to ductility, yield strength and UTS across a range of process variables, including hatch spacing, layer thickness and build orientation (two orientations are considered: horizontal and vertical). The CPFE-predicted effect of build orientation is shown to be consistent with experimental test results. The present methodology has allowed identification of optimal hatch spacing and layer thickness for a given laser spot size, power and scanning speed. CPFE modelling of AM melt pool texture is shown to be key to successful prediction of the effects of process variables on mechanical behaviour.

Guidewires are critically important components in medical implant and device delivery systems and their desired clinical performance or "steerability" requires a good torque response, i.e., the rotation of the (distal) guidewire tip should follow exactly the (proximal) applied input rotation. However, guidewires can suffer from phenomena known as lag (tip rotation is significantly less than the input rotation) and whip (the lag is suddenly recovered). Nitinol is a common guidewire material that can give superior performance but has been known to exhibit lag and whip depending on the specific material formulation and processing route. In this study, the torsional response of Nitinol guidewires is investigated using computational modelling (the finite element method) and analytical approaches, with a view to gaining a fundamental understanding of the mechanisms behind the lag and whip phenomena and how these relate to the specific material properties of a range of Nitinol variants and the geometrical configuration of the wire during torsion. An idealised vascular geometry is considered; this consists of a curved section and a straight section of varying length where the curved section is representative of geometries that are encountered in tortuous path navigation in the vasculature.The results capture the experimentally observed phenomena and reveal the relationship between material properties and guidewire performance. The stress state in the wire, which is dictated by the stress plateaus in the Nitinol material response, leads to the generation of a net moment within the wire which requires net work to be done during rotation of the wire. Analysis of idealised hypothetical materials show that the transformation strain is also an important parameter. The analysis reduced the performance of the guidewire material to a single metric that is given in terms of the energy dissipated during transformation, i.e., the area of the hysteresis loop. The results show that the combination of torsion and the bending of the wire in the curved path are critically important in the generation of lag and whip, and that both are accentuated by increasing the length of the straight section once the phenomena are active.

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (M pro ) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long‐term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1‐SLX4 structure‐specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2‐MSH6), MutSβ (MSH2‐MSH3), and MutLα (MLH1‐PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic‐lethal interaction with promising applications in precision cancer therapy.

COPYRIGHT © 2022 Turchi, Pawelczak, Weinfeld and McHugh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 27 September 2022 DOI 10.3389/fonc.2022.1023500

BackgroundCongenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate.MethodsGenetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.ResultsWe identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.ConclusionStability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

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.