A genome-wide portrait of pervasive drug contaminants.

Using a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.

DNA interaction, in vivo and in vitro cytotoxicity, reactive oxygen species, lipid peroxidation of -N, S donor Re(I) metal complexes.

N, S donor ligands (L1-L5){L1-L5 = 1,5-bis(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole (L1), 1-(4-bromophenyl)-5-(4-chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole (L2), 5-(4-chlorophenyl)-3-(thiophen-2-yl)-1-(p-tolyl)-4,5-dihydro-1H-pyrazole (L3), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole (L4), 5-(4-chlorophenyl)-1-(4-nitrophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole (L5)} were synthesized by Claisen-Schmidt condensation and characterized by spectrometric methods. The complexes (I-V) were synthesized by ligand combination followed by metal chelation. The binding of the rhenium complexes to Herrin sperm DNA was monitored by UV spectroscopy and viscosity measurements. The groove binding was suggested as the most possible mode, and the Kb values of the complexes were calculated. The mode of interaction was furthermore confirmed by molecular docking. Brine shrimp lethality and Saccharomyces cerevisiae cytotoxicity against the eukaryotic and prokaryotic cells showed the toxic nature of the synthesized compounds. All compounds were found active against S. cerevisiae, which was confirmed by increased ROS production, and DNA damage as compared to untreated yeast cell culture. The oxidative harm to cell structures was affirmed by lipid peroxidation. An antimicrobial study was carried out by estimating minimum inhibitory concentration against two Gram-positive and three Gram-negative bacteria. All complexes show good antiproliferative activity against the HCT 116 cell line. All synthesized complexes are biologically more active than the corresponding ligands.

MutSα deficiency increases tolerance to DNA damage in yeast lacking postreplication repair.

By combining mutations in DNA repair genes, important and unexpected interactions between different repair pathways can be discovered. In this study, we identified a novel link between mismatch repair (MMR) genes and postreplication repair (PRR) in Saccharomyces cerevisiae. Strains lacking Rad5 (HLTF in mammals), a protein important for restarting stalled replication forks in the error-free PRR pathway, were supersensitive to the DNA methylating agent methyl methanesulfonate (MMS). Deletion of the mismatch repair genes, MSH2 or MSH6, which together constitutes the MutSα complex, partially suppressed the MMS super-sensitivity of the rad5Δ strain. Deletion of MSH2 also suppressed the MMS sensitivity of mms2Δ, which acts together with Rad5 in error-free PRR. However, inactivating the mismatch repair genes MSH3 and MLH1 did not suppress rad5Δ, showing that the suppression was specific for disabling MutSα. The partial suppression did not require translesion DNA synthesis (REV1, REV3 or RAD30), base excision repair (MAG1) or homologous recombination (RAD51). Instead, the underlying mechanism was dependent on RAD52 while independent of established pathways involving RAD52, like single-strand annealing and break-induced replication. We propose a Rad5- and Rad51-independent template switch pathway, capable of compensating for the loss of the error-free template-switch subpathway of postreplication repair, triggered by the loss of MutSα.

The in Vitro Antiplasmodial and Antiproliferative Activity of New Ferrocene-Based α-Aminocresols Targeting Hemozoin Inhibition and DNA Interaction.

The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α-aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α-aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and -resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple-negative breast cancer cell line. Indication of cross-resistance was absent for the compounds evaluated against the multi-resistant Dd2 strain. Structure-activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α-carbon and NH group of the α-amino-o-cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor-groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

Synergistic effects of ketamine and azole derivatives on Candida spp. resistance to fluconazole.

The emergence of Candida spp. with resistance to antifungal molecules, mainly the azole class, is an increasing complication in hospitals around the globe. Aim: In the present research, we evaluated the synergistic effects of ketamine with two azole derivatives, itraconazole and fluconazole, on strains of Candida spp. to fluconazole. Materials & methods: The drug synergy was evaluated by quantifying the fractional inhibitory concentration index and by fluorescence microscopy and flow cytometry techniques. Results: Our achievements showed a synergistic effect between ketamine in addition to the two antifungal agents (fluconazole and itraconazole) against planktonic cells and biofilms of Candida spp. Conclusion: This combination promoted alteration of membrane integrity, generation of reactive oxygen species, damage to and DNA and externalization of phosphatidylserine.

Shift of Sensitivity in Botrytis cinerea to Benzimidazole Fungicides in Strawberry Greenhouse Ascribing to the Rising-lowering of E198A Subpopulation and its Visual, On-site Monitoring by Loop-mediated Isothermal Amplification.

Grey mold disease results from Botrytis cinerea, a classical "high-risk" plant pathogenic fungus in meaning of resistance development to fungicides, and its management depends largely on the frequent applications of fungicides. The evolution of resistance to benzimidazole chemicals during 2008 and 2016 was monitored continuously in strawberry greenhouses located in Zhejiang province. Results showed that extensive applications of the mixture of carbendazim and diethofencarb caused the rapid spread of Ben MR subpopulation. The withdraw of this mixture lead to the sharply decrease of Ben MR and re-dominance of Ben HR isolates of B. cinerea with the E198A mutation in ß-tubulin gene. The LAMP primers, based on the E198A point mutation, were designed to detect the E198A genotype specifically. HNB (Hydroxynaphthol blue), a metalion indicator, acted as a visual LAMP reaction indicator that turned the violet colored into a sky-blue color. The detection limit of concentration of DNA was 100 × 10-2 ng/µL and this LAMP assay could be applied to detect the E198A genotype with 100% accuracy in strawberry greenhouses of three Province and was more rapid and easier to operate. In summary, we establish a simple and sensitive on-field LAMP assay which can be adopted to determine within 1.5 h whether the benzimidazoles or the mixture of a benzimidazole fungicide and diethofencarb is suitable for management of B. cinerea.

Multifaceted activities of plant gum synthesised platinum nanoparticles: catalytic, peroxidase, PCR enhancing and antioxidant activities.

A single pot, green method for platinum nanoparticles (Pt NP) production was devised with gum ghatti (Anogeissus latifolia). Analytical tools: ultraviolet-visible (UV-vis), dynamic light scattering, zeta potential, transmission electron microscope, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy were employed. Wide continuous UV-vis absorption and black solution colouration proved Pt NP formation. Face-centred cubic crystalline structure of NP was evidenced from XRD. NPs formed were nearly spherical with a mean particle size of 3 nm. NP demonstrated a myriad of properties including catalytic, peroxidase, polymerase chain reaction (PCR) enhancing and antioxidant activities. Catalytic action of NP was probed via NaBH4 reduction of arsenazo-III dye. NP displayed considerable peroxidase activity via catalysis of 3, 3', 5, 5'-tetramethylbenzidine oxidation by H2O2. NP showed exceptional stability towards varying pH (3-11), temperature (25-100°C), salt concentration (0-100 mM) and storage time duration (0-12 months). In comparison with horse radish peroxidase, its applicability as an artificial peroxidase is advantageous. NP caused a two-fold enhancement in PCR yield at 0.4 nM. Also showed significant 1', 1' diphenyl picryl-hydrazyle scavenging (80.1%) at 15 µg/mL. Author envisages that the biogenic Pt NP can be used in a range of biological and environmental applications.

Green tea extract promotes DNA repair in a yeast model.

Green tea polyphenols may protect cells from UV damage through antioxidant activities and by stimulating the removal of damaged or cross-linked DNA. Recently, DNA repair pathways have been predicted as possible targets of epigallocatechin gallate (EGCG)-initiated signaling. However, whether and how green tea polyphenols can promote nucleotide excision repair and homologous recombination in diverse organisms requires further investigation. In this report, we used the budding yeast, Saccharomyces cerevisiae, as a model to investigate the effects of green tea extract on DNA repair pathways. We first showed that green tea extract increased the survival rate and decreased the frequency of mutations in yeast exposed to UVB-irradiation. Furthermore, green tea extract increased the expression of homologous recombination genes, RFA1, RAD51 and RAD52, and nucleotide excision repair genes, RAD4 and RAD14. Importantly, we further used a specific strand invasion assay to show that green tea extract promotes homologous recombination at double-strand breaks. Thus, green tea extract acts to preserve genome stability by activating DNA repair pathways in yeast. Because homologous recombination repair is highly conserved in yeast and humans, this study demonstrates yeast may be a useful platform for future research to investigate the underlying mechanisms of the bioactive compounds in DNA repair.

Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains.

The overproduction of riboflavin (vitamin B2) by Ashbya gossypii, one of the most distinctive traits of this filamentous hemiascomycete, has been proposed to act as an ecological defense mechanism, since it is triggered by environmental stress. The interaction of endogenous riboflavin with light generates reactive oxygen species (ROS) and induces oxidative DNA damage in mammalian cells, but exogenous riboflavin was shown to protect A. gossypii spores against ultraviolet light. Envisioning a better understanding of this biotechnologically relevant trait, here we investigated the putative genotoxic effects associated with the overproduction of riboflavin by A. gossypii. For assessing that we developed the Ashbya Comet Assay, which was able to reproducibly measure oxidative (H2O2/menadione-mediated) and non-oxidative (camptothecin-mediated) DNA damage in A. gossypii. Using this protocol, we determined that exposure to sunlight-mimicking light during growth significantly increased the DNA damage accumulation in riboflavin-overproducing cells, but not in non-overproducing ones. The exposure of overproducing cells to light induced the intracellular accumulation of ROS and increased the production of riboflavin 1.5-fold. These results show that riboflavin-overproducing strains are highly susceptible to photo-induced oxidative DNA damage and draw attention for the importance of controlling the exposure to light of biotechnological riboflavin production processes with A. gossypii.

Doxorubicin induces an extensive transcriptional and metabolic rewiring in yeast cells.

Doxorubicin is one of the most effective chemotherapy drugs used against solid tumors in the treatment of several cancer types. Two different mechanisms, (i) intercalation of doxorubicin into DNA and inhibition of topoisomerase II leading to changes in chromatin structure, (ii) generation of free radicals and oxidative damage to biomolecules, have been proposed to explain the mode of action of this drug in cancer cells. A genome-wide integrative systems biology approach used in the present study to investigate the long-term effect of doxorubicin in Saccharomyces cerevisiae cells indicated the up-regulation of genes involved in response to oxidative stress as well as in Rad53 checkpoint sensing and signaling pathway. Modular analysis of the active sub-network has also revealed the induction of the genes significantly associated with nucleosome assembly/disassembly and DNA repair in response to doxorubicin. Furthermore, an extensive re-wiring of the metabolism was observed. In addition to glycolysis, and sulfate assimilation, several pathways related to ribosome biogenesis/translation, amino acid biosynthesis, nucleotide biosynthesis, de novo IMP biosynthesis and one-carbon metabolism were significantly repressed. Pentose phosphate pathway, MAPK signaling pathway biological processes associated with meiosis and sporulation were found to be induced in response to long-term exposure to doxorubicin in yeast cells.

Oxyresveratrol-induced DNA cleavage triggers apoptotic response in Candida albicans.

Oxyresveratrol is a naturally occurring phytoalexin produced by plants in response to infection. Biological activities of oxyresveratrol have been studied such as antioxidant, anticancer and anti-inflammation. However, further antimicrobial activity and its mechanism need to be investigated. This study exhibited growth inhibition against pathogenic fungi and investigated its mode of action. Oxyresveratrol inflicted cleavage on DNA, leading to G2/M phase arrest. DNA damage by oxyresveratrol was not the result of oxidative stress but it was triggered by direct binding to DNA. Oxyresveratrol-treated cells showed an apoptotic pathway characterized by phosphatidylserine exposure, apoptotic volume decrease and metacaspase activation. Mitochondria-associated apoptotic features also appeared. Oxyresveratrol-induced Ca2+ overload led to mitochondrial membrane depolarization and release of cytochrome c from mitochondria to cytosol. In conclusion, oxyresveratrol with DNA-binding affinity induces DNA cleavage, and eventually leads to mitochondria-mediated apoptosis in Candida albicans.

Novel carbazole-triazole conjugates as DNA-targeting membrane active potentiators against clinical isolated fungi.

A series of carbazole-triazole conjugates were designed, synthesized and characterized by IR, NMR, and HRMS spectra. Biological assay showed that most of the synthesized compounds exhibited moderate and even strong antifungal activities, especially 3,6-dibromocarbazolyl triazole 5d displayed excellent inhibitory efficacy against most of the tested fungal strains (MIC = 2-32 µg/mL) and effectively fungicidal ability towards C. albicans, C. tropicals and C. parapsilosis ATCC 22019 (MFC = 4-8 µg/mL). Its combination use with fluconazole could enhance the antifungal efficacy, and compound 5d also did not obviously trigger the development of resistance in C. albicans even after 10 passages. Preliminary mechanism study revealed that the active molecule 5d could depolarize fungal membrane potential and intercalate into DNA to possibly block DNA replication, thus possibly exhibiting its powerful antifungal abilities. Conjugate 5d could interact with HSA, which was constructive for the further design, modification and screening of drug molecules. Docking investigation demonstrated a non-covalent binding of 5d with CYP51 through hydrogen bond and hydrophobicity. These results strongly suggested that compound 5d could act as a potential template for the development of promising antifungal drugs.

Characterization and antifungal activity against Pestalotiopsis of a fusaricidin-type compound produced by Paenibacillus polymyxa Y-1.

Dendrobium nobile (D. nobile) is a valuable Chinese herbal medicine. The discovery of microbial resources from has provided a wealth of raw materials. Stalk rot, which is caused by Pestalotiopsis, is one of the most serious diseases of D nobile and has resulted in serious losses in production. However, an effective method for the prevention and control of stalk rot remains lacking. In this study, we aimed to identify a biocontrol strain against Pestalotiopsis. We isolated Paenibacillus polymyxa Y-1, an endophytic bacterium, from the stem of D. nobile. Three pairs of active metabolites isolated from this bacterium were identified as fusaricidin compounds. We then investigated the mechanism of fusaricidin compounds on Pestalotiopsis via proteomics. Proteomics data showed that the compounds mainly inhibit energy generation in the respiratory chain and amino acid biosynthesis of Pestalotiopsis.

Bis-guanylhydrazones as efficient anti-Candida compounds through DNA interaction.

Candida spp. are leading causes of opportunistic mycoses, including life-threatening hospital-borne infections, and novel antifungals, preferably aiming targets that have not been used before, are constantly needed. Hydrazone- and guanidine-containing molecules have shown a wide range of biological activities, including recently described excellent antifungal properties. In this study, four bis-guanylhydrazone derivatives (BG1-4) were generated following a previously developed synthetic route. Anti-Candida (two C. albicans, C. glabrata, and C. parapsilosis) minimal inhibitory concentrations (MICs) of bis-guanylhydrazones were between 2 and 15.6 µg/mL. They were also effective against preformed 48-h-old C. albicans biofilms. In vitro DNA interaction, circular dichroism, and molecular docking analysis showed the great ability of these compounds to bind fungal DNA. Competition with DNA-binding stain, exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane, and activation of metacaspases were shown for BG3. This pro-apoptotic effect of BG3 was only partially due to the accumulation of reactive oxygen species in C. albicans, as only twofold MIC and higher concentrations of BG3 caused depolarization of mitochondrial membrane which was accompanied by the decrease of the activity of fungal mitochondrial dehydrogenases, while the activity of oxidative stress response enzymes glutathione reductase and catalase was not significantly affected. BG3 showed synergistic activity with amphotericin B with a fractional inhibitory concentration index of 0.5. It also exerted low cytotoxicity and the ability to inhibit epithelial cell (TR146) invasion and damage by virulent C. albicans SC5314. With further developments, BG3 may further progress in the antifungal pipeline as a DNA-targeting agent.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae.

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation.

SUMO-Targeted DNA Translocase Rrp2 Protects the Genome from Top2-Induced DNA Damage.

The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons. Loss of Rrp2 enhances SUMOylation-dependent ubiquitination and degradation of Top2, which in turn increases DNA damage at sites where Top2-DNA complexes are trapped. Rrp2 possesses SUMO-binding ability and prevents excessive Top2 degradation by competing against the SUMO-targeted ubiquitin ligase (STUbL) for SUMO chain binding and by displacing SUMOylated Top2 from DNA. The budding yeast homolog of Rrp2, Uls1, plays a similar role, indicating that this genome protection mechanism is widely employed, a finding with implications for cancer treatment.

In vitro anti-Candida activity of selective serotonin reuptake inhibitors against fluconazole-resistant strains and their activity against biofilm-forming isolates.

Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24 h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160 µg/mL for fluoxetine, 10-20 µg/mL for sertraline, and 10-100.8 µg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth in vitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis.

Cell cycle-dependent positive and negative functions of Fun30 chromatin remodeler in DNA damage response.

The evolutionally conserved Fun30 chromatin remodeler in Saccharomyces cerevisiae has been shown to contribute to cellular resistance to genotoxic stress inflicted by camptothecin (CPT), methyl methanesulfonate (MMS) and hydroxyurea (HU). Fun30 aids in extensive DNA resection of DNA double stranded break (DSB) ends, which is thought to underlie its role in CPT-resistance. How Fun30 promotes MMS- or HU-resistance has not been resolved. Interestingly, we have recently found Fun30 to also play a negative role in cellular tolerance to MMS and HU in the absence of the Rad5-dependent DNA damage tolerance pathway. In this report, we show that Fun30 acts to down regulate Rad9-dependent DNA damage checkpoint triggered by CPT or MMS, but does not affect Rad9-independent intra-S phase replication checkpoint induced by MMS or HU. These results support the notion that Fun30 contributes to cellular response to DSBs by preventing excessive DNA damage checkpoint activation in addition to its role in facilitating DNA end resection. On the other hand, we present evidence suggesting that Fun30's negative function in MMS- and HU-tolerance in the absence of Rad5 is not related to its regulation of checkpoint activity. Moreover, we find Fun30 to be cell cycle regulated with its abundance peaking in G2/M phase of the cell cycle. Importantly, we demonstrate that artificially restricting Fun30 expression to G2/M does not affect its positive or negative function in genotoxin-resistance, but confining Fun30 to S phase abolishes its functions. These results indicate that both positive and negative functions of Fun30 in DNA damage response occur mainly in G2/M phase.

The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria.

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6µM and when yeast densities ranged between 10(6) to 10(7)CFU/mL and bacterial densities were approximately 10(8)CFU/mL.