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A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

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We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.

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Three human nucleases, SNM1A, SNM1B/Apollo, and SNM1C/Artemis, belong to the SNM1 gene family. These nucleases are involved in various cellular functions, including homologous recombination, nonhomologous end-joining, cell cycle regulation, and telomere maintenance. These three proteins share a similar catalytic domain, which is characterized as a fused metallo-ß-lactamase and a CPSF-Artemis-SNM1-PSO2 domain. SNM1A and SNM1B/Apollo are exonucleases, whereas SNM1C/Artemis is an endonuclease. This review contains a summary of recent research on SNM1's cellular and biochemical functions, as well as structural biology studies. In addition, protein structure prediction by the artificial intelligence program AlphaFold provides a different view of the proteins' non-catalytic domain features, which may be used in combination with current results from X-ray crystallography and cryo-EM to understand their mechanism more clearly.

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Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

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SNM1A is a zinc-dependent nuclease involved in the removal of interstrand crosslink lesions from DNA. Inhibition of interstrand crosslink repair enzymes such as SNM1A is a promising strategy for improving the efficacy of crosslinking chemotherapy drugs. Initial studies have demonstrated the feasibility of developing SNM1A inhibitors, but the full potential of this enzyme as a drug target has yet to be explored. Herein, the synthesis of a family of squaramide- and thiosquaramide-bearing nucleoside derivatives and their evaluation as SNM1A inhibitors is reported. A gel electrophoresis assay was used to identify nucleoside derivatives bearing an N-hydroxysquaramide or squaric acid moiety at the 3'-position, and a thymidine derivative bearing a 5'-thiosquaramide, as candidate SNM1A inhibitors. Quantitative IC50 determination showed that a thymidine derivative bearing a 5'-thiosquaramide was the most potent inhibitor, followed by a thymidine derivative bearing a 3'-squaric acid. UV-Vis titrations were carried out to evaluate the binding of the (thio)squaramides to zinc ions, allowing the order of inhibitory potency to be rationalised. The membrane permeability of the active inhibitors was investigated, with several compounds showing promise for future in vivo applications.

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SNM1A is a nuclease that is implicated in DNA interstrand crosslink repair and, as such, its inhibition is of interest for overcoming resistance to chemotherapeutic crosslinking agents. However, the number and identity of the metal ion(s) in the active site of SNM1A are still unconfirmed, and only a limited number of inhibitors have been reported to date. Herein, we report the synthesis and evaluation of a family of malonate-based modified nucleosides to investigate the optimal positioning of metal-binding groups in nucleoside-derived inhibitors for SNM1A. These compounds include ester, carboxylate and hydroxamic acid malonate derivatives which were installed in the 5'-position or 3'-position of thymidine or as a linkage between two nucleosides. Evaluation as inhibitors of recombinant SNM1A showed that nine of the twelve compounds tested had an inhibitory effect at 1 mM concentration. The most potent compound contains a hydroxamic acid malonate group at the 5'-position. Overall, our studies advance the understanding of requirements for nucleoside-derived inhibitors for SNM1A and indicate that groups containing a negatively charged group in close proximity to a metal chelator, such as hydroxamic acid malonates, are promising structures in the design of inhibitors.

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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.

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DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)- and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

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During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

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Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5'-3' exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit(+) cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.

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A critical step in DNA interstrand cross-link repair is the programmed collapse of replication forks that have stalled at an ICL. This event is regulated by the Fanconi anemia pathway, which suppresses bone marrow failure and cancer. In this perspective, we focus on the structure of forks that have stalled at ICLs, how these structures might be incised by endonucleases, and how incision is regulated by the Fanconi anemia pathway.

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