WRN helicase and mismatch repair complexes independently and synergistically disrupt cruciform DNA structures.

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), MutSß (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.

Neuronal enhancers are hotspots for DNA single-strand break repair.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1.

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

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.

A phosphate binding pocket is a key determinant of exo- versus endo-nucleolytic activity in the SNM1 nuclease family.

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-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.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition.

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-ß-lactamase (MBL) and ß-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 ß-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.

Endovascular versus conventional open surgical repair for thoracoabdominal aortic aneurysms.

BACKGROUND: Thoracoabdominal aortic aneurysms (TAAAs) are a life-threatening condition which remain difficult to treat. Endovascular and open surgical repair (OSR) provide treatment options for patients, however, due to the lack of clinical trials comparing these, the optimum treatment option is unknown. OBJECTIVES: To assess the effectiveness and safety of endovascular repair versus conventional OSR for the treatment of TAAAs. SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Cochrane Vascular Specialised Register, CENTRAL, MEDLINE, Embase, CINAHL and AMED databases and World Health Organization International Clinical Trials Registry Platform and ClinicalTrials.gov trials registers to 26 April 2021. We also searched references of relevant articles retrieved from the electronic search for additional citations. SELECTION CRITERIA: We considered all published and unpublished randomised controlled trials (RCTs) and controlled clinical trials (CCTs) comparing endovascular repair to OSR for TAAAs for inclusion in the review. The main outcomes of interest were prevention of aneurysm rupture (participants without aneurysm rupture up to 5 years from intervention), aneurysm-related mortality (30 days and 12 months), all-cause mortality, spinal cord ischaemia (paraplegia, paraparesis), visceral arterial branch compromise causing mesenteric ischaemia or renal failure, and rate of reintervention. DATA COLLECTION AND ANALYSIS: Two review authors independently screened all titles and abstracts identified from the searches to identify those that met the inclusion criteria. We planned to undertake data collection, risk of bias assessment, and analysis in accordance with Cochrane recommendations. We planned to assess the certainty of the evidence using GRADE. MAIN RESULTS: No RCTs or CCTs met the inclusion criteria for this review. AUTHORS' CONCLUSIONS: Due to the lack of RCTs or CCTs, we were unable to determine the safety and effectiveness of endovascular compared to OSR in patients with TAAAs and are unable to provide any evidence on the optimal surgical intervention for this cohort of patients. High-quality RCTs or CCTs addressing this objective are necessary, however conducting such studies will be logistically and ethically challenging for this life-threatening disease.

Genetic and functional insights into CDA-I prevalence and pathogenesis.

BACKGROUND: Congenital 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. METHODS: Genetic 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. RESULTS: We 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. CONCLUSION: Stability 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.

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

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.

Endovascular versus open surgical repair for complicated chronic Type B aortic dissection.

BACKGROUND: Type B aortic dissection can lead to serious and life-threatening complications such as aortic rupture, stroke, renal failure, and paraplegia, all of which require intervention. Traditionally, these complications have been treated with open surgery. Recently however, endovascular repair has been proposed as an alternative. OBJECTIVES: To assess the effectiveness and safety of thoracic aortic endovascular repair versus open surgical repair for treatment of complicated chronic Type B aortic dissection (CBAD). SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Cochrane Vascular Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, CINAHL, and AMED databases, as well as the World Health Organization International Clinical Trials Registry Platform and ClinicalTrials.gov trials registers, to 2 August 2021. We searched references of relevant articles retrieved through the electronic search for additional citations. SELECTION CRITERIA: We considered all randomised controlled trials (RCTs) and controlled clinical trials (CCTs) assessing the effects of thoracic aortic endovascular repair (TEVAR) versus open surgical repair (OSR) for treatment of complicated chronic Type B aortic dissection (CBAD). Outcomes of interest were mortality (all-cause, dissection-related), neurological sequelae (stroke, spinal cord ischaemia/paresis-paralysis, vertebral insufficiency), morphological outcomes (false lumen thrombosis, progression of dissection, aortic diameters), acute renal failure, ischaemic symptoms (visceral ischaemia, limb ischaemia), re-intervention, and health-related quality of life. DATA COLLECTION AND ANALYSIS: Two review authors independently screened all titles and abstracts identified by the searches to identify those that met the inclusion criteria. From title and abstract screening, we did not identify any trials (RCTs or CCTs) that required full-text assessment. We planned to undertake data collection and analysis in accordance with recommendations described in the Cochrane Handbook for Systematic Reviews of Interventions. We planned to assess the certainty of evidence using GRADE. MAIN RESULTS: We did not identify any trials (RCTs or CCTs) that met the inclusion criteria for this review. AUTHORS' CONCLUSIONS: Due to lack of RCTs or CCTs investigating the effectiveness and safety of TEVAR compared to OSR for patients with complicated CBAD, we are unable to provide any evidence to inform decision-making on the optimal intervention for these patients. High-quality RCTs or CCTs addressing this objective are necessary. However, conducting such studies will be challenging for this life-threatening disease.

Microhomologies are prevalent at Cas9-induced larger deletions.

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

The Chemical Biology of Human Metallo-ß-Lactamase Fold Proteins.

The αßßα metallo ß-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all ß-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

A hydroxamic-acid-containing nucleoside inhibits DNA repair nuclease SNM1A.

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.

Human SNM1A and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 5'-3' exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replication-associated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells.

The scaling of population persistence with carrying capacity does not asymptote in populations of a fish experiencing extreme climate variability.

Despite growing concerns regarding increasing frequency of extreme climate events and declining population sizes, the influence of environmental stochasticity on the relationship between population carrying capacity and time-to-extinction has received little empirical attention. While time-to-extinction increases exponentially with carrying capacity in constant environments, theoretical models suggest increasing environmental stochasticity causes asymptotic scaling, thus making minimum viable carrying capacity vastly uncertain in variable environments. Using empirical estimates of environmental stochasticity in fish metapopulations, we showed that increasing environmental stochasticity resulting from extreme droughts was insufficient to create asymptotic scaling of time-to-extinction with carrying capacity in local populations as predicted by theory. Local time-to-extinction increased with carrying capacity due to declining sensitivity to demographic stochasticity, and the slope of this relationship declined significantly as environmental stochasticity increased. However, recent 1 in 25 yr extreme droughts were insufficient to extirpate populations with large carrying capacity. Consequently, large populations may be more resilient to environmental stochasticity than previously thought. The lack of carrying capacity-related asymptotes in persistence under extreme climate variability reveals how small populations affected by habitat loss or overharvesting, may be disproportionately threatened by increases in extreme climate events with global warming.

The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities.

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-ß-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-ß-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

CSB interacts with SNM1A and promotes DNA interstrand crosslink processing.

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Riparian vegetation as an indicator of riparian condition: Detecting departures from historic condition across the North American West.

Floodplain riparian ecosystems support unique vegetation communities and high biodiversity relative to terrestrial landscapes. Accordingly, estimating riparian ecosystem health across landscapes is critical for sustainable river management. However, methods that identify local riparian vegetation condition, an effective proxy for riparian health, have not been applied across broad, regional extents. Here we present an index to assess reach-scale (500 m segment) riparian vegetation condition across entire drainage networks within large, physiographically-diverse regions. We estimated riparian vegetation condition for 53,250 km of perennial streams and rivers, 25,685 km in Utah, and 27,565 km in twelve watersheds of the interior Columbia River Basin (CRB), USA. We used nationally available, existing land cover classification derived from 30 m Landsat imagery (LANDFIRE EVT) and a modeled estimate of pre-European settlement land cover (LANDFIRE BpS). The index characterizes riparian vegetation condition as the ratio of existing native riparian vegetation cover to pre-European settlement riparian vegetation cover at a given reach. Roughly 62% of Utah and 48% of CRB watersheds showed significant (>33%) to large (>66%) departure from historic condition. Riparian vegetation change was predominantly caused by human land-use impacts (development and agriculture), or vegetation change (native riparian to invasive or upland vegetation types) that likely resulted from flow and disturbance regime alteration. Through comparisons to ground-based classification results, we estimate the existing vegetation component of the index to be 85% accurate. Our assessments yielded riparian condition maps that will help resource managers better prioritize sites and treatments for reach-scale conservation and restoration activities.