Mitochondrial DNA topoisomerase I of Saccharomyces cerevisiae

The radiation protective effect of thiol compounds is unequivocal and their use is only limited by their toxic effects. We used the principle of alpha alkylation, which renders amino acids unmetabolizable, to reduce the toxicity of homocysteine. This product, alpha-methyl-homocysteine thio-lactone, was tested for toxicity and radiation protective effect along with known protectors L-cysteine, cysteamine and WR 1065 in cell culture using V79-4 Chinese hamster lung cells. The three-day growth curve assays, useful to measure overall effects on cell growth, revealed lowest toxicity for alpha-methyl-homocysteine thiolactone (GL-2). Clonogenic survival tests, used to evaluate the retention of reproductive integrity, were carried out and revealed that GL-2 had no adverse effects in this test system. Radiation protection tests showed that GL-2 exhibited protective activity against radiation induced lethality above that seen with cysteine and cysteamine, but below WR 1065. However, GL-2 showed little or no negative effects toward the cell itself, in direct contrast to WR 1065. Our findings show a potentially important tool and principle to reduce toxicity of radiation protectors with analogous structures.     

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.     

Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I

The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.     

DNA sequence analysis of spontaneous mutagenesis in Saccharomyces cerevisiae

To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively. Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis. In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae. The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis.     

The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae

All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures. rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.     

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome

OBJECTIVES: To report the features of malignancies responsible for a chest wall mass and involving the sternum, the sternocostal and/or sternoclavicular joints, the chondrocostal junction and/or the adjacent soft tissues. METHODS: The medical records of patients with a chest wall mass due to malignant disease were reviewed retrospectively. The following data were abstracted from each record: characteristics of the pain and mass, constitutional symptoms, physical findings, laboratory test results, findings from imaging studies (plain radiographs, computed tomography and magnetic resonance imaging of the chest, radionuclide bone scan), histologic features of the biopsy specimen from the chest wall mass and origin of the mass. RESULTS: Seven men and three women with a mean age of 53.1 years were included in the study. A single patient had a history of malignant disease (lymphoma); in the remaining nine patients the chest wall mass was the first manifestation of the malignancy. All ten patients had pain with a mixed time pattern. The mass was located on the sternum in half the patients and in a parasternal location in the other half. Erythrocyte sedimentation rate elevation was found in seven patients, an increased serum level of lactate dehydrogenase in one and a monoclonal immunoglobulin in three. Sternal lesions were visible on plain radiographs in four patients. Computed tomography of the chest consistently disclosed sternal or sternocostal lytic lesions with spread to the adjacent soft tissues; in five cases, enlarged lymph nodes were visible in the anterior part of the mediastinum. Magnetic resonance imaging of the chest did not add to the information provided by computed tomography. Radionuclide uptake on the bone scan was increased, decreased, or normal at the site of the lesion. The cause was Hodgkin's disease in two cases, non-Hodgkin's lymphoma in three, metastatic bone disease in two (from an adenocarcinoma of the lung and a hepatocarcinoma, respectively), multiple myeloma in one, and solitary plasmacytoma in two. CONCLUSION: A chest wall mass can be caused by a known or as yet undiagnosed malignancy. Chest wall involvement due to malignant disease in rare, however. The specific features of sternal metastases, lymphomas involving the sternum, and sternal plasmacytomas are discussed. Nonmalignant chest wall lesions that can manifest as a bulging or swelling of the chest wall are reviewed.     

Nucleotide sequence analysis of the CDNA for rat DNA topoisomerase II

CDNA clones encoding the rat DNA topoisomerase II were isolated from rat testis CDNA library using a DNA probe synthesized by two sequential nested PCRs. The nucleotide sequence of the entire coding region and its deduced 1526 amino acid sequence showed that 80% nucleotides and 89% amino acids were identical with human HeLa DNA topoisomerase II gene (hTOP2). Approximately 1100 amino acids at the N-terminus shows 96.5% sequence identity, but C-terminus has only 65% homology. Rat DNA topoisomerase II gene (rTOP2) contains three functional domains responsible for ATPase activity, break-reunion activity, and complex stability and DNA binding activity like other eukaryotic TOP2. It also contains two putative nuclear targeting sequences and a leucine zipper motif and has highly charged species specific sequences at the C-terminus.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4 gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.     

ALL-1 gene rearrangements in DNA topoisomerase II inhibitor-related leukemia in children

We examined clinical, morphologic, and cytogenetic features and ALL-1 (MLL, Htrxl, HRX) gene rearrangements in 17 cases of secondary leukemia that occurred 11 months to 9 years from diagnoses of primary cancers in children who received topoisomerase II inhibitors or developed secondary leukemias typical of those associated with this therapy. Primary diagnoses included nine solid tumors and eight leukemias. Ten secondary leukemias were acute myeloid leukemia (AML), one was of mixed lineage, two were acute lymphoblastic leukemia (ALL), and four presented as myelodysplasia. Of 15 cases with 11q23 involvement, 11 (73%) were cytogenetically identifiable; four cases had molecular rearrangement only. By Southern blot, rearrangements within the ALL-1 gene were similar to sporadic cases. The results of this analysis suggest the following: (1) In most pediatric cases of topoisomerase II inhibitor-associated leukemia, there is disruption of the breakpoint cluster region of the ALL-1 gene at chromosomal band 11q23. (2) Exposure histories vary in secondary 11q23 leukemia, as the only topoisomerase II inhibitor was dactinomycin in one case, and, in another case, no topoisomerase II inhibitor was administered. (3) There is clinical, morphologic, cytogenetic, and molecular heterogeneity in pediatric secondary 11q23 leukemia. (4) There are some survivors of pediatric secondary 11q23 leukemia, but the outcome is most often fatal.     

Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted DNA damage

Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a 'step-arrest' mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells.     

Unrepaired heteroduplex DNA in Saccharomyces cerevisiae is decreased in RAD1 RAD52-independent recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

A protein-mediated mechanism for the DNA sequence-specific action of topoisomerase II poisons

Chemical agents able to interfere with DNA topoisomerases are widespread in nature, and some of them have outstanding therapeutic efficacy in human cancer and infectious diseases. DNA topoisomerases are essential enzymes that govern DNA topology during fundamental nuclear metabolic processes. Topoisomerase-interfering compounds can be divided into two general categories based on the mechanism of drug action: poisons and catalytic inhibitors. In past years, investigations of the DNA sequence selectivity of topoisomerase II poisons have identified structural and molecular determinants of drug activity, and indicated that the drug receptor is likely to be at the protein-DNA interface. Moreover, the available results indicate that the biologically relevant DNA-binding activity of topoisomerase poisons is basically protein-mediated and this is discussed in this issue by Giovanni Capranico and colleagues. This suggests that topoisomerase poisons may represent a useful paradigm for small compounds able to bind to protein-DNA interfaces in a site-selective manner, thus increasing the affinity of DNA-binding proteins for specific genomic sites.     

Genetic analysis of default mating behavior in Saccharomyces cerevisiae

Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEA2, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating.     

Interaction between the MEC1-dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality.     

Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor. The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.     

Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae

Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the beta-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100-500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b.     

Insulin signaling in the yeast Saccharomyces cerevisiae. 1. Stimulation of glucose metabolism and Snf1 kinase by human insulin

Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.     

A family of hexosephosphate mutases in Saccharomyces cerevisiae

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Topical reversion at the HIS1 locus of Saccharomyces cerevisiae. A tale of three mutants

Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself. Thirteen mutations of his1-1 now have been identified with respect to base sequence. Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, née his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, née his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, née his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion. Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's. Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype. Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis. Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.