Pathways and signatures of mutagenesis at targeted DNA nicks.

Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of 'hourglass' profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

POLQ suppresses interhomolog recombination and loss of heterozygosity at targeted DNA breaks.

Interhomolog recombination (IHR) occurs spontaneously in somatic human cells at frequencies that are low but sufficient to ameliorate some genetic diseases caused by heterozygous mutations or autosomal dominant mutations. Here we demonstrate that DNA nicks or double-strand breaks (DSBs) targeted by CRISPR-Cas9 to both homologs can stimulate IHR and associated copy-neutral loss of heterozygosity (cnLOH) in human cells. The frequency of IHR is 10-fold lower at nicks than at DSBs, but cnLOH is evident in a greater fraction of recombinants. IHR at DSBs occurs predominantly via reciprocal end joining. At DSBs, depletion of POLQ caused a dramatic increase in IHR and in the fraction of recombinants exhibiting cnLOH, suggesting that POLQ promotes end joining in cis, which limits breaks available for recombination in trans These results define conditions that may produce cnLOH as a mutagenic signature in cancer and may, conversely, promote therapeutic correction of both compound heterozygous and dominant negative mutations associated with genetic disease.

Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

Initiation of homologous recombination at DNA nicks.

Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3'-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

Increased levels of RECQ5 shift DNA repair from canonical to alternative pathways.

RECQ5 (RECQL5) is one of several human helicases that dissociates RAD51-DNA filaments. The gene that encodes RECQ5 is frequently amplified in human tumors, but it is not known whether amplification correlates with increased gene expression, or how increased RECQ5 levels affect DNA repair at nicks and double-strand breaks. Here, we address these questions. We show that RECQ5 gene amplification correlates with increased gene expression in human tumors, by in silico analysis of over 9000 individual tumors representing 32 tumor types in the TCGA dataset. We demonstrate that, at double-strand breaks, increased RECQ5 levels inhibited canonical homology-directed repair (HDR) by double-stranded DNA donors, phenocopying the effect of BRCA deficiency. Conversely, at nicks, increased RECQ5 levels stimulated 'alternative' HDR by single-stranded DNA donors, which is normally suppressed by RAD51; this was accompanied by stimulation of mutagenic end-joining. Even modest changes (2-fold) in RECQ5 levels caused significant dysregulation of repair, especially HDR. These results suggest that in some tumors, RECQ5 gene amplification may have profound consequences for genomic instability.

A Genetic Screen Identifies a Critical Role for the WDR81-WDR91 Complex in the Trafficking and Degradation of Tetherin.

Tetherin (BST2/CD317) is a viral restriction factor that anchors enveloped viruses to host cells and limits viral spread. The HIV-1 Vpu accessory protein counteracts tetherin by decreasing its cell surface expression and targeting it for ubiquitin-dependent endolysosomal degradation. Although the Vpu-mediated downregulation of tetherin has been extensively studied, the molecular details are not completely elucidated. We therefore used a forward genetic screen in human haploid KBM7 cells to identify novel genes required for tetherin trafficking. Our screen identified WDR81 as a novel gene required for tetherin trafficking and degradation in both the presence and absence of Vpu. WDR81 is a BEACH-domain containing protein that is also required for the degradation of EGF-stimulated epidermal growth factor receptor (EGFR) and functions in a complex with the WDR91 protein. In the absence of WDR81 the endolysosomal compartment appears swollen, with enlarged early and late endosomes and reduced delivery of endocytosed dextran to cathepsin-active lysosomes. Our data suggest a role for the WDR81-WDR91 complex in the fusion of endolysosomal compartments and the absence of WDR81 leads to impaired receptor trafficking and degradation.

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed.

Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.

DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

Targeted gene therapies: tools, applications, optimization.

Many devastating human diseases are caused by mutations in a single gene that prevent a somatic cell from carrying out its essential functions, or by genetic changes acquired as a result of infectious disease or in the course of cell transformation. Targeted gene therapies have emerged as potential strategies for treatment of such diseases. These therapies depend upon rare-cutting endonucleases to cleave at specific sites in or near disease genes. Targeted gene correction provides a template for homology-directed repair, enabling the cell's own repair pathways to erase the mutation and replace it with the correct sequence. Targeted gene disruption ablates the disease gene, disabling its function. Gene targeting can also promote other kinds of genome engineering, including mutation, insertion, or gene deletion. Targeted gene therapies present significant advantages compared to approaches to gene therapy that depend upon delivery of stably expressing transgenes. Recent progress has been fueled by advances in nuclease discovery and design, and by new strategies that maximize efficiency of targeting and minimize off-target damage. Future progress will build on deeper mechanistic understanding of critical factors and pathways.

Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease.

Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at approximately 1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.

Suicide Risk Among Military Veterans in the Southwestern United States Before and During the COVID-19 Pandemic.

INTRODUCTION: Military Veterans have an increased risk of suicide compared to the general population, but less is known about changes in risk with the onset of the COVID-19 pandemic, or whether any changes have been moderated by psychiatric or demographic factors. The primary objective was to test the hypothesis that the likelihood of suicide attempt or death by suicide was stable during the first year of the pandemic versus the preceding year for the full sample. A second objective was to test the hypothesis that, in contrast, risk increased for Veteran subgroups characterized by traditional risk factors (e.g., psychiatric diagnosis). MATERIALS AND METHODS: We extracted electronic health record data for 771,570 Veterans who received one or more health care visits between March 13, 2019, and March 12, 2021, at eight VA hospitals across the southwestern United States. Primary outcome measures were suicide attempts and deaths by suicide. Predictor variables included psychiatric diagnoses and demographic factors. RESULTS: Multivariable models indicated that the odds of death by suicide did not change during the first year of the COVID-19 pandemic, while the odds of making a suicide attempt declined. Veterans treated for major depression were at heightened risk for attempting suicide in both years, but the association was smaller during the pandemic than the year prior. In contrast, the relative risk of attempt for Veterans who were never married and Veterans treated for a non-alcohol, non-opioid substance-use disorder increased during the pandemic. CONCLUSIONS AND RELEVANCE: The findings suggest that the pandemic has not led to an increase in suicidal behavior, which is consistent with other studies, although the degree of decline varied across diagnostic and demographic groups. Further longitudinal research is needed to evaluate whether the prolonged nature of COVID-19 may lead to changes in risk over time.

Rec25 and Rec27, novel linear-element components, link cohesin to meiotic DNA breakage and recombination.

Meiosis is a specialized nuclear division by which sexually reproducing diploid organisms generate haploid gametes. Recombination between homologous chromosomes facilitates accurate meiotic chromosome segregation and is initiated by DNA double-strand breaks (DSBs) made by the conserved topoisomerase-like protein Spo11 (Rec12 in fission yeast), but DSBs are not evenly distributed across the genome. In Schizosaccharomyces pombe, proteinaceous structures known as linear elements (LinEs) are formed during meiotic prophase. The meiosis-specific cohesin subunits Rec8 and Rec11 are essential for DSB formation in some regions of the genome, as well as for formation of LinEs or the related synaptonemal complex (SC) in other eukaryotes. Proteins required for DSB formation decorate LinEs, and mutants lacking Rec10, a major component of LinEs, are completely defective for recombination. Although recombination may occur in the context of LinEs, it is not well understood how Rec10 is loaded onto chromosomes. We describe two novel components of LinEs in fission yeast, Rec25 and Rec27. Comparisons of rec25Delta, rec27Delta, and rec10Delta mutants suggest multiple pathways to load Rec10. In the major pathway, Rec10 is loaded, together with Rec25 and Rec27, in a Rec8-dependent manner with subsequent region-specific effects on recombination.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.

A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events.

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.

Assaying Repair at DNA Nicks.

Nicks are the most common form of DNA damage, but they have only recently been shown to initiate damage that requires repair. Analysis of the pathways of nick repair in human cells has benefited from the development of enzymes that target nicks to specific sites in the genome and of reporters that enable rapid analysis of homology-directed repair and mutagenic end joining. Nicks undergo efficient repair by single-stranded oligonucleotide donors complementary to either the nicked or intact DNA strand, via pathways that are normally suppressed by RAD51. Here we discuss the details of reporter assays that take advantage of the convenience and sensitivity of flow cytometry to analyze pathways of repair at targeted DNA nicks. These assays are readily carried out in 96-well format cell culture plates, enabling mechanistic questions to be addressed by determining the contributions of specific factors by depletion and/or ectopic expression.

The meiotic bouquet promotes homolog interactions and restricts ectopic recombination in Schizosaccharomyces pombe.

Chromosome architecture undergoes extensive, programmed changes as cells enter meiosis. A highly conserved change is the clustering of telomeres at the nuclear periphery to form the "bouquet" configuration. In the fission yeast Schizosaccharomyces pombe the bouquet and associated nuclear movement facilitate initial interactions between homologs. We show that Bqt2, a meiosis-specific protein required for bouquet formation, is required for wild-type levels of homolog pairing and meiotic allelic recombination. Both gene conversion and crossing over are reduced and exhibit negative interference in bqt2Delta mutants, reflecting reduced homolog pairing. While both the bouquet and nuclear movement promote pairing, only the bouquet restricts ectopic recombination (that between dispersed repetitive DNA). We discuss mechanisms by which the bouquet may prevent deleterious translocations by restricting ectopic recombination.

The Rhodococcus equi virulence protein VapA disrupts endolysosome function and stimulates lysosome biogenesis.

Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence-associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria.