Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/ PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/ Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/ Apollo À/À ATM À/À MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5 0 -3 0 exonuclease function of SNM1B/Apollo in the generation of 3 0 single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway. The EMBO Journal (2010) IntroductionMammalian telomeres consist of TTAGGG repetitive sequences that terminate in a 3 0 single-stranded (ss) G-rich overhang. Telomeres are bound and stabilized by a number of telomere-specific-binding proteins that form a core complex termed shelterin that protects telomeres from inappropriately activating the DNA damage response (DDR) (Palm and de Lange, 2008). Three sequence-specific DNA-binding proteins are recruited to chromosomal ends: the duplex telomerebinding proteins TRF1 and TRF2/RAP1, and the ss TTAGGG repeat-binding protein POT1. These proteins are interconnected by the adapter proteins TIN2 and TPP1. Telomeres rendered dysfunctional by the removal of TRF2/RAP1 activate ATM and are repaired by the non-homologous endjoining (NHEJ) pathway, whereas removal of the POT1-TPP1 complex activates NHEJ-mediated repair that requires ATR (Wu et al, 2006;Denchi and de Lange, 2007;Guo et al, 2007;Deng et al, 2009).Emerging evidence suggests that the core shelterin complex is insufficient for complete chromosomal end protection. Rather, accessory proteins that interact with the shelterin complex are also essential for telomere stability. One such protein is SNM1B/Apollo, a member of a small gene family that also includes SNM1A and SNMIC/Artemis. All three proteins share sequence similarity to the yeast interstrand crosslink (ICL) repair protein PSO2/SNM1 (Dronkert et al, 2000). These proteins are characterized by a conserved metallo-b-lactamase-fold and an appended b-CPSF-ArtemisSnm1-Pso2 (CASP) domain that together imparts 5 0 exonuclease function (Callebaut et al, 2002;Poinsignon et al, 2004;Lenain et al, 2006). SNM1A localizes to ionizing radiation (IR)-induced DNA breaks (Richie et al...

BackgroundWe analyzed data from the baseline assessment of a large intervention project to describe typical handwashing practices in rural Bangladesh, and compare measures of hand cleanliness with household characteristics.MethodsWe randomly selected 100 villages from 36 districts in rural Bangladesh. Field workers identified 17 eligible households per village using systematic sampling. Field workers conducted 5-hour structured observations in 1000 households, and a cross-sectional assessment in 1692 households that included spot checks, an evaluation of hand cleanliness and a request that residents demonstrate their usual handwashing practices after defecation.ResultsAlthough 47% of caregivers reported and 51% demonstrated washing both hands with soap after defecation, in structured observation, only 33% of caregivers and 14% of all persons observed washed both hands with soap after defecation. Less than 1% used soap and water for handwashing before eating and/or feeding a child. More commonly people washed their hands only with water, 23% after defecation and 5% before eating. Spot checks during the cross sectional survey classified 930 caregivers (55%) and 453 children (28%) as having clean appearing hands. In multivariate analysis economic status and water available at handwashing locations were significantly associated with hand cleanliness among both caregivers and children.ConclusionsA minority of rural Bangladeshi residents washed both hands with soap at key handwashing times, though rinsing hands with only water was more common. To realize the health benefits of handwashing, efforts to improve handwashing in these communities should target adding soap to current hand rinsing practices.

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We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37°C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.Cryptococcus neoformans is a basidiomycetous fungus and a major human pathogen. This pathogenic fungus has a wide human host range by producing infections in both immunocompromised and immunocompetent hosts (6). Understanding the mechanisms of how this encapsulated yeast can so effectively attack a susceptible host has received renewed attention as the infrastructure for molecular biology of this yeast has matured. Several phenotypic factors in the virulence composite, such as formation of a polysaccharide capsule (7), melanin production (45), urease synthesis (11), phospholipase secretion (10), mannose production (46), high-temperature growth (28), and several signaling molecules and pathways for these factors (44,28

Summary We identified UHRF1 as a binding factor for DNA interstrand crosslink (ICL) lesions through affinity purification of ICL-recognition activities. UHRF1 is recruited to DNA lesions in vivo and binds directly to ICL-containing DNA. UHRF1-deficient cells display increased sensitivity to a variety of DNA damages. We found that loss of UHRF1 led to retarded lesion processing and reduced recruitment of ICL repair nucleases to the site of DNA damage. UHRF1 interacts physically with both ERCC1 and MUS81, two nucleases involved in the repair of ICL lesions. Depletion of both UHRF1 and components of the Fanconi anemia pathway resulted in increased DNA damage sensitivity compared to defect of each mechanism alone. These results suggest that UHRF1 promotes recruitment of lesion-processing activities via its DNA damage recognition affinity and functions as a nuclease recruitment scaffold in parallel to the Fanconi anemia pathway.

Cell division cycle 5-like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre-messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell-cycle checkpoint kinase ataxia-telangiectasia and Rad3-related (ATR). Depletion of Cdc5L by RNA-mediated interference methods results in a defective S-phase cell-cycle checkpoint and cellular sensitivity in response to replication-fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR-binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L-depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR-mediated cell-cycle checkpoint in response to genotoxic agents.

Spindle poisons represent an important class of anticancer drugs that act by interfering with microtubule polymerization and dynamics and thereby induce mitotic checkpoints and apoptosis. Here we show that mammalian SNM1 functions in an early mitotic stress checkpoint that is distinct from the well-characterized spindle checkpoint that regulates the metaphase-to-anaphase transition. Specifically, we found that compared to wild-type cells, Snm1-deficient mouse embryonic fibroblasts exposed to spindle poisons exhibited elevated levels of micronucleus formation, decreased mitotic delay, a failure to arrest in mitosis prior to chromosome condensation, supernumerary centrosomes, and decreased viability. In addition, we show that both Snm1 and 53BP1, previously shown to interact, coimmunoprecipitate with components of the anaphase-promoting complex (APC)/cyclosome. These findings suggest that Snm1 is a component of a mitotic stress checkpoint that negatively targets the APC prior to chromosome condensation.

The archetypical member of the SNM1 gene family was discovered 30 years ago in the budding yeast Saccharomyces cerevisiae. This small but ubiquitous gene family is characterized by metallo-β-lactamase and β-CASP domains, which together have been demonstrated to comprise a nuclease activity. Three mammalian members of this family, SNM1A, SNM1B/Apollo and Artemis, have been demonstrated to play surprisingly divergent roles in cellular metabolism. These pathways include variable (diversity) joining recombination, nonhomologous end-joining of double-strand breaks, DNA damage and mitotic cell cycle checkpoints, telomere maintenance and protein ubiquitination. Not all of these functions are consistent with a model in which these proteins act only as nucleases, and indicate that the SNM1 gene family encodes multifunctional products that can act in diverse biochemical pathways. In this article we discuss the various functions of SNM1A, SNM1B/Apollo and Artemis. KeywordsApollo; Artemis; cell cycle; phosphorylation; SNM1; telomere Approximately 30 years ago, independent genetic screens conducted in Saccharomyces cerevisiae for hypersensitivity to the DNA interstrand cross-linking (ICL) agents nitrogen mustard and psoralen identified the founding member of a small but ubiquitous gene family referred to as sensitivity to nitrogen mustard (SNM1) or sensitivity to psoralen (PSO2) [1][2][3][4][5][6][7]. An intriguing aspect of the yeast SNM1/PSO2 mutants is that they were singularly hypersensitive to bi-and polyfunctional alkylating compounds, while exhibiting an essentially wild-type level of sensitivity to monofunctional alkylating agents, ionizing radiation (IR) or UV. This unique phenotype led a number of laboratories to search for mammalian homologs of SNM1/PSO2, and five such genes were identified, including SNM1A, SNM1B/Apollo, Artemis, CPSF73 and ELAC2. Both CPSF73 and ELAC2 have roles in RNA processing [8][9][10], while the other three members have various roles in DNA metabolism and cellular stress responses. It is the functions of SNM1A, SNM1B/Apollo and Artemis that are the subject of this article.Molecular cloning of the yeast SNM1/PSO2 gene and its mammalian homologs demonstrated that the common features of this family are a metallo-β-lactamase (MBL) fold and an appended