Role of Saccharomyces cerevisiae chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo

In vitro, the protein complex Chromatin Assembly Factor-I (CAF-I) from human or yeast cells deposits histones onto DNA templates after replication. In Saccharomyces cerevisiae, the CAC1, CAC2, and CAC3 genes encode the three CAF-I subunits. Deletion of any of the three CAC genes reduces telomeric gene silencing and confers an increase in sensitivity to killing by ultraviolet (UV) radiation. We used double and triple mutants involving cac1Delta and yeast repair gene mutations to show that deletion of the CAC1 gene increases the UV sensitivity of cells mutant in genes from each of the known DNA repair epistasis groups. For example, double mutants involving cac1Delta and excision repair gene deletions rad1Delta or rad14Delta showed increased UV sensitivity, as did double mutants involving cac1Delta and deletions of members of the RAD51 recombinational repair group. cac1Delta also increased the UV sensitivity of strains with defects in either the error-prone (rev3Delta) or error-free (pol30-46) branches of RAD6-mediated postreplicative DNA repair but did not substantially increase the sensitivity of strains carrying null mutations in the RAD6 or RAD18 genes. Deletion of CAC1 also increased the UV sensitivity and rate of UV-induced mutagenesis in rad5Delta mutants, as has been observed for mutants defective in error-free postreplicative repair. Together, these data suggest that CAF-I has a role in error-free postreplicative damage repair and may also have an auxiliary role in other repair mechanisms. Like the CAC genes, RAD6 is also required for gene silencing at telomeres. We find an increased loss of telomeric gene silencing in rad6Delta cac1Delta and rad18Delta cac1Delta double mutants, suggesting that CAF-I and multiple factors in the postreplicative repair pathway influence chromosome structure.     

Sir3p domains involved in the initiation of telomeric silencing in Saccharomyces cerevisiae

Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3(N205) mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD "active" site is under both positive and negative control mediated by multiple Sir3p domains.     

Histone modification governs the cell cycle regulation of a replication-independent chromatin assembly pathway in Saccharomyces cerevisiae

We describe a replication-independent, cell cycle-regulated chromatin assembly pathway in budding yeast. The activity of this pathway is low in S phase extracts but is very high in G2, M, and G1 cell extracts, with peak activity in late M/early G1. The cell cycle regulation of this pathway requires a specific pattern of posttranslational modification of histones H3 and/or H4, which is distinct for H3/H4 present in S phase versus M and G1 phase cell extracts. Histone H3/H4 modification is therefore important for the reciprocal control of replication-dependent and -independent chromatin assembly pathways during the cell cycle.     

Hyperthermic modification of radiation induced gene conversion in Saccharomyces cerevisiae

Effect of pre- and post-irradiation hyperthermia on the induction of gene conversion (non-reciprocal recombination) in diploid yeast cells was investigated. Post irradiation heat treatment does not significantly modify the frequency of gene conversion. Both low-level (51 degrees C-10 min) and lethal (51 degrees C, 40 min) pre irradiation heat treatments enhanced the gene conversion frequency by 17-49%. There was no quantitative correlation between the observed enhancement and the radiation dose. These results suggest that hyperthermia given prior to radiation not only sensitizes the cells to killing but also increases their convertogenic response. It has been suggested that recombination may be involved in carcinogenesis and tumour promotion. Based on these observations along with other reports, wherein hyperthermia was shown to modify the carcinogenic effects in animals as well as the genetic effects of radiation in vitro, it is possible to suggest that hyperthermia may have a potential to increase the second primary cancers, especially in long term survivors. Certain differences in the mechanism of interaction between radiation and heat in the pre- irradiation and post irradiation modalities appear likely.     

Isocitrate lyase localisation in Saccharomyces cerevisiae cells

The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.     

Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae

We recently showed the involvement of the L-arginine/nitric oxide (NO) pathway in acid-induced duodenal mucosal bicarbonate secretion in rats. The aim of the present study was to confirm this observation in pigs by direct measurements of NO production. Experiments were performed on 16 anaesthetized pigs of both sexes treated with guanethidine (6 mg kg-1, intravenously). A duodenal segment, devoid of pancreaticobiliary influxes, was perfused with saline and the duodenal mucosal bicarbonate secretion was calculated from continuous measurements of pH and PCO2. The perfusate contents of NO and its oxidative product nitrite were determined by chemiluminescence, after reduction of nitrite to NO. Luminal acidification with 30 mM hydrochloric acid increased the output of bicarbonate as well as NO to the perfusate, by 195 +/- 45% and 106 +/- 10%, respectively. These responses to acid were markedly inhibited by adding the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.3 mM) to the perfusate. The inhibitory effect of L-NMMA could be reversed by administration of L-arginine (3 mM). The study presents simultaneous measurements of bicarbonate and NO outputs to a duodenal luminal perfusate. The results strongly support the view that the L-arginine/NO pathway is involved in the acid-induced duodenal mucosal bicarbonate secretory response.     

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.     

Enhanced sensitivity of ubiquinone-deficient mutants of Saccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids

Coenzyme Q (ubiquinone or Q) plays a well known electron transport function in the respiratory chain, and recent evidence suggests that the reduced form of ubiquinone (QH2) may play a second role as a potent lipid-soluble antioxidant. To probe the function of QH2 as an antioxidant in vivo, we have made use of a Q-deficient strain of Saccharomyces cerevisiae harboring a deletion in the COQ3 gene [Clarke, C. F., Williams, W. & Teruya, J. H. (1991) J. Biol. Chem. 266, 16636-16644]. Q-deficient yeast and the wild-type parental strain were subjected to treatment with polyunsaturated fatty acids, which are prone to autoxidation and breakdown into toxic products. In this study we find that Q-deficient yeast are hypersensitive to the autoxidation products of linolenic acid and other polyunsaturated fatty acids. In contrast, the monounsaturated oleic acid, which is resistant to autoxidative breakdown, has no effect. The hypersensitivity of the coq3delta strains can be prevented by the presence of the COQ3 gene on a single copy plasmid, indicating that the sensitive phenotype results solely from the inability to produce Q. As a result of polyunsaturated fatty acid treatment, there is a marked elevation of lipid hydroperoxides in the coq3 mutant as compared with either wild-type or respiratory-deficient control strains. The hypersensitivity of the Q-deficient mutant can be rescued by the addition of butylated hydroxytoluene, alpha-tocopherol, or trolox, an aqueous soluble vitamin E analog. The results indicate that autoxidation products of polyunsaturated fatty acids mediate the cell killing and that QH2 plays an important role in vivo in protecting eukaryotic cells from these products.     

Synthesis and intracellular transport of aminoglycerophospholipids in permeabilized cells of the yeast, Saccharomyces cerevisiae

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.     

Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione

Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level. However, the mechanism of cytotoxicity of this compound still remains controversial. This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione. Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells. GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains. These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells.     

Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.     

Recombination and the progression of meiosis in Saccharomyces cerevisiae

Recombination is an essential part of meiosis: in almost all organisms, including Saccharomyces cerevisiae, proper chromosome segregation and the viability of meiotic products is dependent upon normal levels of recombination. In this article we examine the kinetics of the meiotic divisions in four mutants defective in the initiation of recombination. We find that mutations in any of three Early Exchange genes (REC104, REC114 or REC102) confer a phenotype in which the reductional division occurs earlier than in an isogenic wild-type diploid. We also present data confirming previous reports that strains with a mutation in the Early Exchange gene. MEI4 undergo the first division at about the same time as wild-type cells. The rec104 mutation is epistatic to the mei4 mutation for the timing of the first division. These observations suggest a possible relationship between the initiation of recombination and the timing of the reductional division. These data also allow these four Early Exchange genes examined to be distinguished in terms of their role in coordinating recombination with the reductional division.     

Similar processes mediate glycopeptide export from the endoplasmic reticulum in mammalian cells and Saccharomyces cerevisiae

Glycopeptides are transported from the lumen of the yeast endoplasmic reticulum (ER) to the cytosol and in contrast to secretory proteins do not enter ER-to-Golgi transport vesicles. In a cell-free system, this process is ATP- and cytosol-dependent. While yeast cytosol promotes the export of glycopeptides from mammalian ER in vitro, glycopeptide release cannot be detected in the presence of mammalian cytosol. We demonstrate that this is due to an N-glycanase activity in mammalian cytosol rather than lack of glycopeptide transport activity in mammalian microsomes. Monitoring the amount of glycopeptide enclosed in ER membranes we show the cytosol- and ATP-dependent release of glycopeptide from mammalian microsomes. The fact that glycopeptide export can be achieved with ER and cytosol derived from heterologous sources indicates that glycopeptide export from the ER is an important process conserved during evolution.