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Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest. [ABSTRACT FROM AUTHOR]

BacA proteins play key roles in the chronic intracellular infections of Sinorhizobium meliloti, Brucella abortus and Mycobacterium tuberculosis within their respective hosts. S. meliloti, B. abortus and M. tuberculosis BacA-deficient mutants have increased resistance to the thiazole-modified peptide bleomycin. BacA has been previously hypothesized, but not experimentally verified, to be involved in bleomycin uptake. In this paper, we show that a BacA-dependent mechanism is the major route of bleomycin internalization in S. meliloti. We also determined that the B. abortus and S. meliloti BacA proteins are functional homologues and that the B. abortus BacA protein is involved in the uptake of both bleomycin and proline-rich peptides. Our findings also provide evidence that there is a second, BacA-independent minor mechanism for bleomycin internalization in S. meliloti. We determined that the BacA-dependent and -independent mechanisms of bleomycin uptake are energy-dependent, consistent with both mechanisms of bleomycin uptake involving transport systems. DOI 10.1099/mic.0.039909-0

Saccharomyces cerevisiae Opt2 is a member of the oligopeptide transporter family that was initially identified to transport tetra- and pentapeptides. Mutants deleted for the OPT2 gene exhibit no growth defects under normal culture conditions. We identified OPT2 from a high-throughput screen that when deleted results in mutants that displayed sensitivity to the anticancer agent bleomycin. The opt2Δ mutant was also reisolated in two additional genome-wide screens designed to identify mutants that are sensitive to the immunosuppressant rapamycin and the divalent metal ion zinc. However, t he role of Opt2 in protecting cells against these agents was not investigated. Herein, we show that opt2A mutants are also sensitive to a wide variety of toxic agents that are typically detoxified by the vacuoles. Mutants lacking two other related oligop eptide transporters, Opt1 and Yg1114w, showed no significant sensitivities to these drugs, indicating a specific role for Opt2 in drug detoxification. The sensitivities of the opt2Δ mutants were not related to an increased drug uptake but rather to the presence of several small vesicles instead of a functional large vacuole. We propose that Opt2 has a novel function involving the fusion of vesicles to form a mature vacuole. Key words: bleomycin, polyamines, divalent ions, sensitivity, vital dye, vacuolar morphology. Resume: Chez Saccaromyces cerevisiae, Opt2 est un membre de la famille des transporteurs d'oligopeptides d'abord identifie dans le transport des tetra- et des pentapeptides. Les mutants de deletion du gene OPT2 ne presentent pas de defauts de croissance en conditions de culture normales. Nous avons identifie OPT2 lors d'un criblage a haut debit qui, lorsque supprime, produit des mutants sensibles a la bleomycine, un agent anticancereux. Le mutant opt2Δ a aussi ete isole de nouveau, lors de deux criblages additionnels sur tout le genome ayant pour but d'identifier des mutants sensibles a l'immunosuppresseur rapamycine et au zinc, un ion metallique divalent. Cependant, le role de Opt2 dans la protection des cellules contre ces agents n'avait pas ete examine. Nous montrons ici que les mutants opt2Δ sont aussi sensibles a une grande variete d'agents toxiques typiquement detoxiques par les vacuoles. Les mutants deficients en deux autres transporteurs d'oligopeptides, Opt1 et Yg1114w, ne montraient aucune sensibilite significative envers ces drogues, indiquant que Opt2 joue un role specifique dans la detoxication des drogues. La sensibilite des mutants opt2Δ n'etait pas reliee a une augmentation de la captation de ces drogues, mais a la presence de plusieurs petites vesicules a la place d'une grande vacuole fonctionn elle. Nous proposons que Opt2 possede une nouvelle fonction impliquant la fusion des vesicules pour former une vacuole mature. Mots-cles : bleomycine, polyamines, ions divalents, sensibilite, colorant vital, morphologie vacuolaire. [Traduit par la Redaction], Introduction All organisms have devised a mechanism involving cytoplasmic membrane transporters to specifically take up small peptides in the range of two to six amino acid residues (Hauser et al. [...]

We show that mutants lacking either the phosphatase activator Rrd1 or the phosphatase Pph3 are resistant to rapamycin and that double mutants exhibit a synergistic response. This phenotype could be related to an inability of the mutants to degrade RNA polymerase II, leading to transcription of critical genes that sustain growth.

A yeast DNA-repair-deficient mutant devoid of apurinic/apyrimidinic (AP) endonuclease/3'-diesterase activities is used to express the Caenorhabditis elegans EXO-3 as a functionally active Gst-fusion protein. Results reveal that the purified Gst-EXO-3 possesses both AP endonuclease and 3'-diesterase activities and that a variant D190A completely lacks these activities.

Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. In yeast, rapamycin inhibits the TORC1 kinase signaling pathway causing rapid alteration in gene expression and ultimately cell cycle arrest in G1 through mechanisms that are not fully understood. Herein, we screened a histone mutant collection and report that one of the mutants, H2B R95A, is strikingly resistant to rapamycin due to a defective cell cycle arrest. We show that the H2B R95A causes defects in the expression of a subset of genes of the pheromone pathway required for α factor-induced G1 arrest. The expression of the STE5 gene and its encoded scaffold protein Ste5, required for the sequential activation of the MAPKs of the pheromone pathway, is greatly reduced in the H2B R95A mutant. Similar to the H2B R95A mutant, cells devoid of Ste5 are also resistant to rapamycin. Rapamycin-induced G1 arrest does not involve detectable phosphorylation of the MAPKs, Kss1, and Fus3, as reported for α factor-induced G1 arrest. However, we observed a sharp induction of the G1 cyclin Cln2 (~ 3- to 4-fold) in the ste5Δ mutant within 30 min of exposure to rapamycin. Our data provide a new insight whereby rapamycin signaling via the Torc1 kinase may exploit the pheromone pathway to arrest cells in the G1 phase. [ABSTRACT FROM AUTHOR]

DNA 3'-phosphatase mediates the removal of 3'-phosphate ends generated by the Escherichia coli fpg apurinic/apyrimidinic (AP) lyase following the incision of AP site. The protein does not cleave DNA bearing AP sites, indicating that it is not an AP endonuclease or AP lyase.

A study was conducted to analyze amino acid substitutions that hinder the ability of Escherichia coli endo IV, a bifunctional DNA repair protein, to carry out its biological function in repairing damaged DNA. The parent AB1157 and the mutant BW528 were the bacterial strains utilized in the study. Results indicated that thapyrimidinic endonuclease and the 3'-diesterase activities of endo IV correlate with a single active site that is remote from the DNA binding domain.