Mechanisms of genotoxicity and proteotoxicity induced by the metalloids arsenic and antimony.

Arsenic and antimony are metalloids with profound effects on biological systems and human health. Both elements are toxic to cells and organisms, and exposure is associated with several pathological conditions including cancer and neurodegenerative disorders. At the same time, arsenic- and antimony-containing compounds are used in the treatment of multiple diseases. Although these metalloids can both cause and cure disease, their modes of molecular action are incompletely understood. The past decades have seen major advances in our understanding of arsenic and antimony toxicity, emphasizing genotoxicity and proteotoxicity as key contributors to pathogenesis. In this review, we highlight mechanisms by which arsenic and antimony cause toxicity, focusing on their genotoxic and proteotoxic effects. The mechanisms used by cells to maintain proteostasis during metalloid exposure are also described. Furthermore, we address how metalloid-induced proteotoxicity may promote neurodegenerative disease and how genotoxicity and proteotoxicity may be interrelated and together contribute to proteinopathies. A deeper understanding of cellular toxicity and response mechanisms and their links to pathogenesis may promote the development of strategies for both disease prevention and treatment.

ISW1a modulates cohesin distribution in centromeric and pericentromeric regions.

Cohesin is a highly conserved, multiprotein complex whose canonical function is to hold sister chromatids together to ensure accurate chromosome segregation. Cohesin association with chromatin relies on the Scc2-Scc4 cohesin loading complex that enables cohesin ring opening and topological entrapment of sister DNAs. To better understand how sister chromatid cohesion is regulated, we performed a proteomic screen in budding yeast that identified the Isw1 chromatin remodeler as a cohesin binding partner. In addition, we found that Isw1 also interacts with Scc2-Scc4. Lack of Isw1 protein, the Ioc3 subunit of ISW1a or Isw1 chromatin remodeling activity resulted in increased accumulation of cohesin at centromeres and pericentromeres, suggesting that ISW1a may promote efficient translocation of cohesin from the centromeric site of loading to neighboring regions. Consistent with the role of ISW1a in the chromatin organization of centromeric regions, Isw1 was found to be recruited to centromeres. In its absence we observed changes in the nucleosomal landscape at centromeres and pericentromeres. Finally, we discovered that upon loss of RSC functionality, ISW1a activity leads to reduced cohesin binding and cohesion defect. Taken together, our results support the notion of a key role of chromatin remodelers in the regulation of cohesin distribution on chromosomes.

Comparison of Antibacterial Mode of Action of Silver Ions and Silver Nanoformulations With Different Physico-Chemical Properties: Experimental and Computational Studies.

The aim of this study was to compare the antibacterial mode of action of silver ions (Ag+) and selected silver nanoformulations against E. coli strains (E. coli J53, Escherichia coli BW25113 and its derivatives: Δ ompA, Δ ompC, Δ ompF, Δ ompR, ompRG596AcusSG1130A, cusSG1130A). In this research we used various experimental methods and techniques such as determination of the minimal inhibitory concentration, flow cytometry, scanning electron microscopy, circular dichroism as well as computational methods of theoretical chemistry. Thanks to the processing of bacteria and silver samples (ions and nanoformulations), we were able to determine the bacterial sensitivity to silver samples, detect reactive oxygen species (ROS) in the bacterial cells, visualize the interaction of silver samples with the bacterial cells, and identify their interactions with proteins. Differences between the mode of action of silver ions and nanoformulations and the action of nanoformulations themselves were revealed. Based on the results of computational methods, we proposed an explanation of the differences in silver-outer protein interaction between silver ions and metallic silver; in general, the Ag0 complexes exhibit weaker interaction than Ag+ ones. Moreover, we identified two gutter-like areas of the inner layer of the ion channel: one more effective, with oxygen-rich side chains; and another one less effective, with nitrogen-rich side chains.

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions, Telomere Dysfunction and Inhibition of DNA Repair.

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.

The Anticancer Drug 3-Bromopyruvate Induces DNA Damage Potentially Through Reactive Oxygen Species in Yeast and in Human Cancer Cells.

3-bromopyruvate (3-BP) is a small molecule with anticancer and antimicrobial activities. 3-BP is taken up selectively by cancer cells' mono-carboxylate transporters (MCTs), which are highly overexpressed by many cancers. When 3-BP enters cancer cells it inactivates several glycolytic and mitochondrial enzymes, leading to ATP depletion and the generation of reactive oxygen species. While mechanisms of 3-BP uptake and its influence on cell metabolism are well understood, the impact of 3-BP at certain concentrations on DNA integrity has never been investigated in detail. Here we have collected several lines of evidence suggesting that 3-BP induces DNA damage probably as a result of ROS generation, in both yeast and human cancer cells, when its concentration is sufficiently low and most cells are still viable. We also demonstrate that in yeast 3-BP treatment leads to generation of DNA double-strand breaks only in S-phase of the cell cycle, possibly as a result of oxidative DNA damage. This leads to DNA damage, checkpoint activation and focal accumulation of the DNA response proteins. Interestingly, in human cancer cells exposure to 3-BP also induces DNA breaks that trigger H2A.X phosphorylation. Our current data shed new light on the mechanisms by which a sufficiently low concentration of 3-BP can induce cytotoxicity at the DNA level, a finding that might be important for the future design of anticancer therapies.

The Emerging Role of Cohesin in the DNA Damage Response.

Faithful transmission of genetic material is crucial for all organisms since changes in genetic information may result in genomic instability that causes developmental disorders and cancers. Thus, understanding the mechanisms that preserve genome integrity is of fundamental importance. Cohesin is a multiprotein complex whose canonical function is to hold sister chromatids together from S-phase until the onset of anaphase to ensure the equal division of chromosomes. However, recent research points to a crucial function of cohesin in the DNA damage response (DDR). In this review, we summarize recent advances in the understanding of cohesin function in DNA damage signaling and repair. First, we focus on cohesin architecture and molecular mechanisms that govern sister chromatid cohesion. Next, we briefly characterize the main DDR pathways. Finally, we describe mechanisms that determine cohesin accumulation at DNA damage sites and discuss possible roles of cohesin in DDR.

Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin.

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.

New insights into cohesin loading.

Cohesin is a conserved, ring-shaped protein complex that encircles sister chromatids and ensures correct chromosome segregation during mitosis and meiosis. It also plays a crucial role in the regulation of gene expression, DNA condensation, and DNA repair through both non-homologous end joining and homologous recombination. Cohesins are spatiotemporally regulated by the Scc2-Scc4 complex which facilitates cohesin loading onto chromatin at specific chromosomal sites. Over the last few years, much attention has been paid to cohesin and cohesin loader as it became clear that even minor disruptions of these complexes may lead to developmental disorders and cancers. Here we summarize recent developments in the structure of Scc2-Scc4 complex, cohesin loading process, and mediators that determine the Scc2-Scc4 binding patterns to chromatin.

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast.

Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin.

DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae.

DNA damage tolerance and homologous recombination pathways function to bypass replication-blocking lesions and ensure completion of DNA replication. However, inappropriate activation of these pathways may lead to increased mutagenesis or formation of deleterious recombination intermediates, often leading to cell death or cancer formation in higher organisms. Post-translational modifications of PCNA regulate the choice of repair pathways at replication forks. Its monoubiquitination favors translesion synthesis, while polyubiquitination stimulates template switching. Srs2 helicase binds to small ubiquitin-related modifier (SUMO)-modified PCNA to suppress a subset of Rad51-dependent homologous recombination. Conversely, SUMOylation of Srs2 attenuates its interaction with PCNA Sgs1 helicase and Mus81 endonuclease are crucial for disentanglement of repair intermediates at the replication fork. Deletion of both genes is lethal and can be rescued by inactivation of Rad51-dependent homologous recombination. Here we show that Saccharomyces cerevisiae Uls1, a member of the Swi2/Snf2 family of ATPases and a SUMO-targeted ubiquitin ligase, physically interacts with both PCNA and Srs2, and promotes Srs2 binding to PCNA by downregulating Srs2-SUMO levels at replication forks. We also identify deletion of ULS1 as a suppressor of mus81Δ sgs1Δ synthetic lethality and hypothesize that uls1Δ mutation results in a partial inactivation of the homologous recombination pathway, detrimental in cells devoid of both Sgs1 and Mus81 We thus propose that Uls1 contributes to the pathway where intermediates generated at replication forks are dismantled by Srs2 bound to SUMO-PCNA. Upon ULS1 deletion, accumulating Srs2-SUMO-unable to bind PCNA-takes part in an alternative PCNA-independent recombination repair salvage pathway(s).

Swi2/Snf2-like protein Uls1 functions in the Sgs1-dependent pathway of maintenance of rDNA stability and alleviation of replication stress.

The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.

Oxidative stress and replication-independent DNA breakage induced by arsenic in Saccharomyces cerevisiae.

Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70-Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70-Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.

The Swi2-Snf2-like protein Uls1 is involved in replication stress response.

The Saccharomyces cerevisiae Uls1 belongs to the Swi2-Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here, we examine a physiological role of Uls1 and report for the first time its involvement in response to replication stress. We found that deletion of ULS1 in cells lacking RAD52 caused a synthetic growth defect accompanied by prolonged S phase and aberrant cell morphology. uls1Δ also progressed slower through S phase upon MMS treatment and took longer to resolve replication intermediates during recovery. This suggests an important function for Uls1 during replication stress. Consistently, cells lacking Uls1 and endonuclease Mus81 were more sensitive to HU, MMS and CPT than single mus81Δ. Interestingly, deletion of ULS1 attenuated replication stress-related defects in sgs1Δ, such as sensitivity to HU and MMS while increasing the level of PCNA ubiquitination and Rad53 phosphorylation. Importantly, Uls1 interactions with Mus81 and Sgs1 were dependent on its helicase domain. We propose that Uls1 directs a subset of DNA structures arising during replication into the Sgs1-dependent pathway facilitating S phase progression. Thus, in the absence of Uls1 other modes of replication fork processing and repair are employed.

[Pathways of arsenic uptake in prokaryotic and eukaryotic cells]. / Mechanizmy transportu nieorganicznych zwiazków arsenu do komórek prokariotycznych i eukariotycznych.

Mechanisms of arsenic uptake and detoxification are present in all studied organisms. These mechanisms are considerably well described in unicellular organisms such as bacterium Escherichia coli and baker's yeast Saccharomyces cerevisiae, still leaving much to be revealed in multicellular organisms. Full identification of arsenic uptake and detoxification is of great importance. This knowledge can be very helpful in improving effectiveness of arsenic-containing drugs used in chemotherapy of parasitoses as well as in treatment of acute promielyocytic leukemia. Increased proficiency of bioremediation of arsenic-contaminated soils can be obtained by using plants hyperaccumulating arsenic. This kind of plants can be engineered by modulating expression levels of genes encoding arsenic transporters. The same technique may be used to decrease levels of accumulated arsenic in crops. The aim of this paper is to review current knowledge about systems of arsenic uptake in every studied organism--from bacteria to human.