Spontaneous Frameshift Mutations in Saccharomyces cerevisiae: Accumulation During DNA Replication and Removal by Proofreading and Mismatch Repair Activities

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Christopher N Greene ◽  
Sue Jinks-Robertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Wild Type Strain ◽  
Dna Polymerases ◽  
Wild Type ◽  
Triple Mutant ◽  
Repair Capacity ◽  
Frameshift Mutations ◽  
Dna Polymerization ◽  
In The Wild

Abstract The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2ΔBgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases δ and ε (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 535-542 ◽  
Author(s):  
B A Kunz ◽  
M G Peters ◽  
S E Kohalmi ◽  
J D Armstrong ◽  
M Glattke ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutator Phenotype ◽  
In The Wild ◽  
Single Base Pair ◽  
Almost All

Abstract Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (2) ◽  
pp. 1278-1292 ◽  
Author(s):  
C Mezard ◽  
A Nicolas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Type Strain ◽  
Wild Type Strain ◽  
Double Strand Breaks ◽  
Linear Plasmid ◽  
Wild Type ◽  
Dsb Repair ◽  
Strand Breaks ◽  
In The Wild

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

1994 ◽  
Vol 14 (2) ◽  
pp. 1278-1292
Author(s):  
C Mezard ◽  
A Nicolas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Type Strain ◽  
Wild Type Strain ◽  
Double Strand Breaks ◽  
Linear Plasmid ◽  
Wild Type ◽  
Dsb Repair ◽  
Strand Breaks ◽  
In The Wild

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.

scholarly journals Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

1999 ◽  
Vol 181 (2) ◽  
pp. 396-400 ◽  
Author(s):  
H. H. W. Silljé ◽  
J. W. G. Paalman ◽  
E. G. ter Schure ◽  
S. Q. B. Olsthoorn ◽  
A. J. Verkleij ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Growth Rates ◽  
Type Strain ◽  
Cell Cycle Progression ◽  
Wild Type Strain ◽  
Carbon Limitation ◽  
Wild Type ◽  
Cycle Progression ◽  
In The Wild

ABSTRACT Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO2production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.

scholarly journals Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 131 (2) ◽  
pp. 261-276 ◽  
Author(s):  
K J Dornfeld ◽  
D M Livingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Inverted Repeat ◽  
Wild Type Strain ◽  
Direct Repeat ◽  
Cell Types ◽  
Wild Type ◽  
Plasmid Recombination ◽  
Intramolecular Recombination ◽  
In The Wild ◽  
Repeat Events

Abstract Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.

scholarly journals Saccharomyces cerevisiae null mutants in glucose phosphorylation: metabolism and invertase expression.

Genetics ◽  
1991 ◽  
Vol 128 (3) ◽  
pp. 521-527 ◽  
Author(s):  
R B Walsh ◽  
D Clifton ◽  
J Horak ◽  
D G Fraenkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Wild Type ◽  
Aerobic Growth ◽  
Triple Mutant ◽  
Hexokinase 2 ◽  
Glucose Flux ◽  
In The Wild ◽  
Glucose Phosphorylation ◽  
Different Strains

Abstract A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism.

scholarly journals Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Dariel Márquez ◽  
Ximena Escalera-Fanjul ◽  
Mohammed el Hafidi ◽  
Beatriz Aguirre-López ◽  
Lina Riego-Ruiz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Alanine Transaminase ◽  
Aminobutyric Acid ◽  
Heat Sensitivity ◽  
Wild Type ◽  
Gaba Shunt ◽  
In The Wild

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

scholarly journals Antioxidant Activity Evaluation of Dietary Flavonoid Hyperoside Using Saccharomyces Cerevisiae as a Model

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 788 ◽  
Author(s):  
Yuting Gao ◽  
Lianying Fang ◽  
Xiangxing Wang ◽  
Ruoni Lan ◽  
Meiyan Wang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxidative Damage ◽  
Cell Viability ◽  
Wild Type Strain ◽  
Wild Type ◽  
Ros Scavenging ◽  
Defence Mechanisms ◽  
Intracellular Ros ◽  
In The Wild ◽  
Dietary Flavonoid

Oxidative stress leads to various diseases, including diabetes, cardiovascular diseases, neurodegenerative diseases, and even cancer. The dietary flavonol glycoside, hyperoside (quercetin-3-O-galactoside), exerts health benefits by preventing oxidative damage. To further understand its antioxidative defence mechanisms, we systemically investigated the regulation of hyperoside on oxidative damage induced by hydrogen peroxide, carbon tetrachloride, and cadmium in Saccharomyces cerevisiae. Hyperoside significantly increased cell viability, decreased lipid peroxidation, and lowered intracellular reactive oxygen species (ROS) levels in the wild-type strain (WT) and mutants gtt1∆ and gtt2∆. However, the strain with ctt1∆ showed variable cell viability and intracellular ROS-scavenging ability in response to the hyperoside treatment upon the stimulation of H2O2 and CCl4. In addition, hyperoside did not confer viability tolerance or intercellular ROS in CdSO4-induced stress to strains of sod1∆ and gsh1∆. The results suggest that the antioxidative reactions of hyperoside in S. cerevisiae depend on the intercellular ROS detoxification system.

Sign in / Sign up

Export Citation Format

Share Document