scholarly journals Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

2012 ◽  
Vol 13 (3) ◽  
pp. 2846-2862 ◽  
Author(s):  
Samuel Saito ◽  
Givaldo Silva ◽  
Regineide Xavier Santos ◽  
Grace Gosmann ◽  
Cristina Pungartnik ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Adducts ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cassia Alata

scholarly journals Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2309
Author(s):  
Stanislav G. Kozmin ◽  
Gregory Eot-Houllier ◽  
Anne Reynaud-Angelin ◽  
Didier Gasparutto ◽  
Evelyne Sage
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Excision Repair ◽  
Cell Extract ◽  
Yeast Saccharomyces Cerevisiae ◽  
Strand Breaks ◽  
High Mutation Frequency ◽  
Base Excision ◽  
Ionizing Radiation Exposure

Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.

scholarly journals Identification of New Markers of Alcohol-Derived DNA Damage in Humans

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 366
Author(s):  
Valeria Guidolin ◽  
Erik S. Carlson ◽  
Andrea Carrà ◽  
Peter W. Villalta ◽  
Laura A. Maertens ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Adducts ◽  
Neutral Loss ◽  
Alcohol Exposure ◽  
Dose Dependence ◽  
Accurate Mass ◽  
Constant Neutral Loss ◽  
Covalent Modifications ◽  
Underlying Mechanisms

Alcohol consumption is a risk factor for the development of several cancers, including those of the head and neck and the esophagus. The underlying mechanisms of alcohol-induced carcinogenesis remain unclear; however, at these sites, alcohol-derived acetaldehyde seems to play a major role. By reacting with DNA, acetaldehyde generates covalent modifications (adducts) that can lead to mutations. Previous studies have shown a dose dependence between levels of a major acetaldehyde-derived DNA adduct and alcohol exposure in oral-cell DNA. The goal of this study was to optimize a mass spectrometry (MS)-based DNA adductomic approach to screen for all acetaldehyde-derived DNA adducts to more comprehensively characterize the genotoxic effects of acetaldehyde in humans. A high-resolution/-accurate-mass data-dependent constant-neutral-loss-MS3 methodology was developed to profile acetaldehyde-DNA adducts in purified DNA. This resulted in the identification of 22 DNA adducts. In addition to the expected N2-ethyldeoxyguanosine (after NaBH3CN reduction), two previously unreported adducts showed prominent signals in the mass spectra. MSn fragmentation spectra and accurate mass were used to hypothesize the structure of the two new adducts, which were then identified as N6-ethyldeoxyadenosine and N4-ethyldeoxycytidine by comparison with synthesized standards. These adducts were quantified in DNA isolated from oral cells collected from volunteers exposed to alcohol, revealing a significant increase after the exposure. In addition, 17 of the adducts identified in vitro were detected in these samples confirming our ability to more comprehensively characterize the DNA damage deriving from alcohol exposures.

scholarly journals The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (4) ◽  
pp. 832-835 ◽  
Author(s):  
Terri S. Rice ◽  
Min Ding ◽  
David S. Pederson ◽  
Nicholas H. Heintz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Origin Recognition Complex ◽  
Nuclear Division ◽  
Permissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
M Phase ◽  
And Migration ◽  
Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

scholarly journals The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 2719-2727 ◽  
Author(s):  
S Silve ◽  
P Leplatois ◽  
A Josse ◽  
P H Dupuy ◽  
C Lanau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Proliferation ◽  
Gene Product ◽  
In Vitro Assays ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isomerase Activity ◽  
Encoding Gene ◽  
Resistant Mutants

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

scholarly journals Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1805-1812 ◽  
Author(s):  
J Zhu ◽  
R H Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Aberrations ◽  
Topoisomerase I ◽  
Wild Type Strain ◽  
Hot Spot ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

scholarly journals Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (4) ◽  
pp. 1371-1377 ◽  
Author(s):  
T Toda ◽  
S Cameron ◽  
P Sass ◽  
M Zoller ◽  
J D Scott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Carbon Sources ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Regulatory Subunits ◽  
Dependent Protein

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

1990 ◽  
Vol 10 (11) ◽  
pp. 5679-5687
Author(s):  
C K Barlowe ◽  
D R Appling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Point Mutations ◽  
Gene Replacement ◽  
Molecular Genetic Analysis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function

1987 ◽  
Vol 7 (7) ◽  
pp. 2344-2351
Author(s):  
R J Deschenes ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Membrane Fraction ◽  
Carbon Sources ◽  
Fatty Acyl ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fatty Acyl Moiety ◽  
Fatty Acylation ◽  
Absolute Requirement

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.

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