Two Dot1 isoforms in Saccharomyces cerevisiae as a result of leaky scanning by the ribosome

Dot1 is a conserved histone methyltransferase that methylates histone H3 on lysine 79. We previously observed that in Saccharomyces cerevisiae, a single DOT1 gene encodes two Dot1 protein species. Here, we show that the relative abundance of the two isoforms changed under nutrient-limiting conditions. A mutagenesis approach showed that the two Dot1 isoforms are produced from two alternative translation start sites as a result of leaky scanning by the ribosome. The leaky scanning was not affected by the 5′- or 3′-untranslated regions of DOT1, indicating that translation initiation is determined by the DOT1 coding sequence. Construction of yeast strains expressing either one of the isoforms showed that both were sufficient for Dot1’s role in global H3K79 methylation and telomeric gene silencing. However, the absence of the long isoform of Dot1 altered the resistance of yeast cells to the chitin-binding drug Calcofluor White, suggesting that the two Dot1 isoforms have a differential function in cell wall biogenesis.     

Role of Dot1 in the response to alkylating DNA damage in Saccharomyces cerevisiae: regulation of DNA damage tolerance by the error-prone polymerases Polzeta/Rev1

Maintenance of genomic integrity relies on a proper response to DNA injuries integrated by the DNA damage checkpoint; histone modifications play an important role in this response. Dot1 methylates lysine 79 of histone H3. In Saccharomyces cerevisiae, Dot1 is required for the meiotic recombination checkpoint as well as for chromatin silencing and the G(1)/S and intra-S DNA damage checkpoints in vegetative cells. Here, we report the analysis of the function of Dot1 in the response to alkylating damage. Unexpectedly, deletion of DOT1 results in increased resistance to the alkylating agent methyl methanesulfonate (MMS). This phenotype is independent of the dot1 silencing defect and does not result from reduced levels of DNA damage. Deletion of DOT1 partially or totally suppresses the MMS sensitivity of various DNA repair mutants (rad52, rad54, yku80, rad1, rad14, apn1, rad5, rad30). However, the rev1 dot1 and rev3 dot1 mutants show enhanced MMS sensitivity and dot1 does not attenuate the MMS sensitivity of rad52 rev3 or rad52 rev1. In addition, Rev3-dependent MMS-induced mutagenesis is increased in dot1 cells. We propose that Dot1 inhibits translesion synthesis (TLS) by Polzeta/Rev1 and that the MMS resistance observed in the dot1 mutant results from the enhanced TLS activity.     

Effects of dietary selenium on glutathione peroxidase and thioredoxin reductase activity and recovery from cardiac ischemia-reperfusion.

Glutathione peroxidase and thioredoxin reductase are selenocysteine-dependent enzymes that protect against oxidative injury. This study examined the effects of dietary selenium on the activity of these two enzymes in rats, and investigated the ability of selenium to modulate myocardial function post ischemia-reperfusion. Male wistar rats were fed diets containing 0, 50, 240 and 1000 microg/kg sodium selenite for 5 weeks. Langendorff perfused hearts isolated from these rats were subjected to 22.5 min global ischemia and 45 min reperfusion, with functional recovery assessed. Liver samples were collected at the time of sacrifice, and heart and liver tissues assayed for thioredoxin reductase and glutathione peroxidase activity. Selenium deficiency reduced the activity of both glutathione peroxidase and thioredoxin reductase systemically. Hearts from selenium deficient animals were more susceptible to ischemia-reperfusion injury when compared to normal controls (38% recovery of rate pressure product (RPP) vs. 47% recovery of RPP). Selenium supplementation increased the endogenous activity of thioredoxin reductase and glutathione peroxidase and resulted in improved recovery of cardiac function post ischemia reperfusion (57% recovery of RPP). Endogenous activity of glutathione peroxidase and thioredoxin reductase is dependent on an adequate supply of the micronutrient selenium. Reduced activity of these antioxidant enzymes is associated with significant reductions in myocardial function post ischemia-reperfusion.     

Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase

Yeast disruptor of telomeric silencing-1 (DOT1) is involved in gene silencing and in the pachytene checkpoint during meiotic cell cycle. Here we show that the Dot1 protein possesses intrinsic histone methyltransferase (HMT) activity. When compared with Rmt1, another putative yeast HMT, Dot1 shows very distinct substrate specificity. While Rmt1 methylates histone H4, Dot1 targets histone H3. In contrast to Rmt1, which can only modify free histones, Dot1 activity is specific to nucleosomal substrates. This was also confirmed using native chromatin purified from yeast cells. We also demonstrate that, like its mammalian homolog PRMT1, Rmt1 specifically dimethylates an arginine residue at position 3 of histone H4 N-terminal tail. In surprising contrast, methylation by Dot1 occurs in the globular domain of nucleosomal histone H3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis suggests that H3 lysine 79 is trimethylated by Dot1. The intrinsic nucleosomal histone H3 methyltransferase activity of Dot1 is certainly a key aspect of its function in gene silencing at telomeres, most likely by directly modulating chromatin structure and Sir protein localization. In agreement with a role in regulating localization of histone deacetylase complexes like SIR, an increase of bulk histone acetylation is detected in dot1- cells.     

Dot1p modulates silencing in yeast by methylation of the nucleosome core

DOT1 was originally identified as a gene affecting telomeric silencing in S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates approximately 90% of histone H3. In dot1delta cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.     

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.     

Peroxiredoxins in Regulation of MAPK Signalling Pathways; Sensors and Barriers to Signal Transduction

Peroxiredoxins are highly conserved and abundant peroxidases. Although the thioredoxin peroxidase activity of peroxiredoxin (Prx) is important to maintain low levels of endogenous hydrogen peroxide, Prx have also been shown to promote hydrogen peroxide-mediated signalling. Mitogen activated protein kinase (MAPK) signalling pathways mediate cellular responses to a variety of stimuli, including reactive oxygen species (ROS). Here we review the evidence that Prx can act as both sensors and barriers to the activation of MAPK and discuss the underlying mechanisms involved, focusing in particular on the relationship with thioredoxin.     

A second class of peroxidases linked to the trypanothione metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, has three nearly identical genes encoding cysteine homologues of classical selenocysteine-containing glutathione peroxidases. The proteins are expressed in the mammalian and insect stages of the parasite. One of the genes, which contains a mitochondrial as well as a glycosomal targeting signal has been overexpressed. The recombinant T. brucei peroxidase has a high preference for the trypanothione/tryparedoxin couple as electron donor for the reduction of different hydroperoxides but accepts also T. brucei thioredoxin. The apparent rate constants k(2)' for the regeneration of the reduced enzyme are 2 x 10(5) m(-1) s(-1) with tryparedoxin and 5 x 10(3) m(-1) s(-1) with thioredoxin. No saturation kinetics was observed and the rate-limiting step of the overall reaction is reduction of the hydroperoxide. With glutathione, the peroxidase has marginal activity and reduction of the enzymes becomes limiting with a k(2)' value of 3 m (-1) s(-1). The T. brucei peroxidase, in contrast to the related Trypanosoma cruzi enzyme, also accepts hydrogen peroxide as substrate. The catalytic efficiency of the peroxidase studied here is comparable with that of the peroxiredoxin-like tryparedoxin peroxidases, which shows that trypanosomes possess two distinct peroxidase systems both dependent on the unique dithiol trypanothione.     

A putative glutathione peroxidase of Drosophila encodes a thioredoxin peroxidase that provides resistance against oxidative stress but fails to complement a lack of catalase activity

Cellular defense systems against reactive oxygen species (ROS) include thioredoxin reductase (TrxR) and glutathione reductase (GR). They generate sulfhydryl-reducing systems which are coupled to antioxidant enzymes, the thioredoxin and glutathione peroxidases (TPx and GPx). The fruit fly Drosophila lacks a functional GR, suggesting that the thioredoxin system is the major source for recycling glutathione. Whole genome in silico analysis identified two non-selenium containing putative GPx genes. We examined the biochemical characteristics of one of these gene products and found that it lacks GPx activity and functions as a TPx. Transgene-dependent overexpression of the newly identified Glutathione peroxidase homolog with thioredoxin peroxidase activity (Gtpx-1) gene increases resistance to experimentally induced oxidative stress, but does not compensate for the loss of catalase, an enzyme which, like GTPx-1, functions to eliminate hydrogen peroxide. The results suggest that GTPx-1 is part of the Drosophila Trx antioxidant defense system but acts in a genetically distinct pathway or in a different cellular compartment than catalase.     

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica , Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.