scholarly journals Genome-wide analysis of DNA turnover and gene expression in stationary-phase Saccharomyces cerevisiae

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1758-1771 ◽  
Author(s):  
A. de Morgan ◽  
L. Brodsky ◽  
Y. Ronin ◽  
E. Nevo ◽  
A. Korol ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Stationary Phase ◽  
Dna Synthesis ◽  
Exponential Phase ◽  
Yeast Cells ◽  
Genome Wide ◽  
Dna Turnover ◽  
Genomic Scale ◽  
Dna Maintenance

Exponential-phase yeast cells readily enter stationary phase when transferred to fresh, carbon-deficient medium, and can remain fully viable for up to several months. It is known that stationary-phase prokaryotic cells may still synthesize substantial amounts of DNA. Although the basis of this phenomenon remains unclear, this DNA synthesis may be the result of DNA maintenance and repair, recombination, and stress-induced transposition of mobile elements, which may occur in the absence of DNA replication. To the best of our knowledge, the existence of DNA turnover in stationary-phase unicellular eukaryotes remains largely unstudied. By performing cDNA-spotted (i.e. ORF) microarray analysis of stationary cultures of a haploid Saccharomyces cerevisiae strain, we demonstrated on a genomic scale the localization of a DNA-turnover marker [5-bromo-2′-deoxyuridine (BrdU); an analogue of thymidine], indicative of DNA synthesis in discrete, multiple sites across the genome. Exponential-phase cells on the other hand, exhibited a uniform, total genomic DNA synthesis pattern, possibly the result of DNA replication. Interestingly, BrdU-labelled sites exhibited a significant overlap with highly expressed features. We also found that the distribution among chromosomes of BrdU-labelled and expressed features deviates from random distribution; this was also observed for the overlapping set. Ty1 retrotransposon genes were also found to be labelled with BrdU, evidence for transposition during stationary phase; however, they were not significantly expressed. We discuss the relevance and possible connection of these results to DNA repair, mutation and related phenomena in higher eukaryotes.

A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Synthetic Lethality ◽  
Velocity Sedimentation ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

scholarly journals Coupling DNA Replication and Spindle Function in Saccharomyces cerevisiae

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3359
Author(s):  
Dimitris Liakopoulos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Molecular Mechanisms ◽  
Genome Duplication ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Dynamics ◽  
Eukaryotic Organisms ◽  
Spindle Function

In the yeast Saccharomyces cerevisiae DNA replication and spindle assembly can overlap. Therefore, signaling mechanisms modulate spindle dynamics in order to ensure correct timing of chromosome segregation relative to genome duplication, especially when replication is incomplete or the DNA becomes damaged. This review focuses on the molecular mechanisms that coordinate DNA replication and spindle dynamics, as well as on the role of spindle-dependent forces in DNA repair. Understanding the coupling between genome duplication and spindle function in yeast cells can provide important insights into similar processes operating in other eukaryotic organisms, including humans.

scholarly journals Yeast Meiotic Mutants Proficient for the Induction of Ectopic Recombination

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 581-598
Author(s):  
JoAnne Engebrecht ◽  
Sherie Masse ◽  
Luther Davis ◽  
Kristine Rose ◽  
Therese Kessel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Meiotic Division ◽  
Ectopic Recombination ◽  
Meiotic Mutants ◽  
Chromosome Synapsis

Abstract A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.

scholarly journals Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae. Effect of starvation and ageing.

2002 ◽  
Vol 49 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Anna Szkopinska ◽  
Ewa Swiezewska ◽  
Joanna Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Yeast Cells ◽  
Logarithmic Phase ◽  
Cellular Membranes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Chemical Changes ◽  
Physico Chemical ◽  
Long Chain

The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.

Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo

1992 ◽  
Vol 12 (12) ◽  
pp. 5724-5735
Author(s):  
J Miles ◽  
T Formosa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Yeast Cells ◽  
Chromosome Loss ◽  
Dna Metabolism ◽  
Checkpoint Gene ◽  
Dna Polymerase Alpha ◽  
In Cells

Potential DNA replication accessory factors from the yeast Saccharomyces cerevisiae have previously been identified by their ability to bind to DNA polymerase alpha protein affinity matrices (J. Miles and T. Formosa, Proc. Natl. Acad. Sci. USA 89:1276-1280, 1992). We have now used genetic methods to characterize the gene encoding one of these DNA polymerase alpha-binding proteins (POB1) to determine whether it plays a role in DNA replication in vivo. We find that yeast cells lacking POB1 are viable but display a constellation of phenotypes indicating defective DNA metabolism. Populations of cells lacking POB1 accumulate abnormally high numbers of enlarged large-budded cells with a single nucleus at the neck of the bud. The average DNA content in a population of cells lacking POB1 is shifted toward the G2 value. These two phenotypes indicate that while the bulk of DNA replication is completed without POB1, mitosis is delayed. Deleting POB1 also causes elevated levels of both chromosome loss and genetic recombination, enhances the temperature sensitivity of cells with mutant DNA polymerase alpha genes, causes increased sensitivity to UV radiation in cells lacking a functional RAD9 checkpoint gene, and causes an increased probability of death in cells carrying a mutation in the MEC1 checkpoint gene. The sequence of the POB1 gene indicates that it is identical to the CTF4 (CHL15) gene identified previously in screens for mutations that diminish the fidelity of chromosome transmission. These phenotypes are consistent with defective DNA metabolism in cells lacking POB1 and strongly suggest that this DNA polymerase alpha-binding protein plays a role in accurately duplicating the genome in vivo.

scholarly journals Interaction of replication factor Sld3 and histone acetyl transferase Esa1 alleviates gene silencing and promotes the activation of late and dormant replication origins

2020 ◽  
Author(s):  
Seiji Tanaka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Gene Silencing ◽  
Dna Synthesis ◽  
Histone Acetyltransferase ◽  
Replication Origins ◽  
Histone Acetyl Transferase ◽  
Step Process ◽  
Helicase Loading ◽  
Rate Limiting

SUMMARYDNA replication in eukaryotes is a multi-step process that consists of three main reactions: helicase loading (licensing), helicase activation (firing), and nascent DNA synthesis (elongation). Although the contributions of some chromatin regulatory factors in the licensing and elongation reaction have been determined, their functions in the firing reaction remain elusive. In the budding yeast Saccharomyces cerevisiae, Sld3, Sld7, and Cdc45 (3-7-45) are rate-limiting in the firing reaction and simultaneous overexpression of 3-7-45 causes untimely activation of late and dormant replication origins. Here we found that 3-7-45 overexpression not only activated dormant origins in the silenced locus, HMLα, but also exerted an anti-silencing effect at this locus. For these, interaction between Sld3 and Esa1, a conserved histone acetyltransferase, was responsible. Moreover, the Sld3–Esa1 interaction was required for untimely activation of late origins. These results reveal the Sld3–Esa1 interaction as a novel level of regulation in the firing reaction.

scholarly journals The Saccharomyces cerevisiae MUM2 Gene Interacts With the DNA Replication Machinery and Is Required for Meiotic Levels of Double Strand Breaks

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1179-1189 ◽  
Author(s):  
Luther Davis ◽  
Maria Barbera ◽  
Amanda McDonnell ◽  
Katherine McIntyre ◽  
Rolf Sternglanz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Origin Recognition Complex ◽  
Wild Type ◽  
Replication Machinery ◽  
Synthesis Inhibitor ◽  
Strand Breaks ◽  
Meiotic Gene

Abstract The Saccharomyces cerevisiae MUM2 gene is essential for meiotic, but not mitotic, DNA replication and thus sporulation. Genetic interactions between MUM2 and a component of the origin recognition complex and polymerase α-primase suggest that MUM2 influences the function of the DNA replication machinery. Early meiotic gene expression is induced to a much greater extent in mum2 cells than in meiotic cells treated with the DNA synthesis inhibitor hydroxyurea. This result indicates that the mum2 meiotic arrest is downstream of the arrest induced by hydroxyurea and suggests that DNA synthesis is initiated in the mutant. Genetic analyses indicate that the recombination that occurs in mum2 mutants is dependent on the normal recombination machinery and on synaptonemal complex components and therefore is not a consequence of lesions created by incompletely replicated DNA. Both meiotic ectopic and allelic recombination are similarly reduced in the mum2 mutant, and the levels are consistent with the levels of meiosis-specific DSBs that are generated. Cytological analyses of mum2 mutants show that chromosome pairing and synapsis occur, although at reduced levels compared to wild type. Given the near-wild-type levels of meiotic gene expression, pairing, and synapsis, we suggest that the reduction in DNA replication is directly responsible for the reduced level of DSBs and meiotic recombination.

Über den biologischen Einfluß von N-Methyl-N-β-chloräthylhydrazin und seinem Benzaldehydhydrazon auf Hefezellen ( Saccharomyces cerevisiae) / Effect of N-Methyl-N-β-chloroethyl-hydrazine-HCl on the Growth and Multiplication of Yeast Cells (Saccharomyces cerevisiae)

1976 ◽  
Vol 31 (5-6) ◽  
pp. 292-297 ◽  
Author(s):  
R. Braun ◽  
H. Lange
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Yeast Cells

Abstract N-Methyl-N-β-chloroethyl-hydrazine-HCl and its benzaldehydhydrazone inhibit growth and multi­plication of yeast cells. The DNA-synthesis is partially blocked. After removal of the substances the rate of this synthesis is much more increased than that of RNA and protein. The both sub­ stances we tested are more effective than simple alkylhydrazines.

Developmental regulation of a sporulation-specific enzyme activity in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (2) ◽  
pp. 171-178
Author(s):  
M J Clancy ◽  
L M Smith ◽  
P T Magee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Developmental Regulation ◽  
Specific Enzyme ◽  
Rapid Degradation ◽  
Specific Enzyme Activity ◽  
Alpha Glucosidase ◽  
Meiosis I

An alpha-glucosidase activity (SAG) occurs in a/alpha Saccharomyces cerevisiae cells beginning at about 8 to 10 h after the initiation of sporulation. This enzyme is responsible for the rapid degradation of intracellular glycogen which follows the completion of meiosis in these cells. SAG differs from similar activities present in vegetative cells and appears to be a sporulation-specific enzyme. Cells arrested at various stages in sporulation (DNA replication, recombination, meiosis I, and meiosis II) were examined for SAG activity; the results show that SAG appearance depends on DNA synthesis and some recombination events but not on the meiotic divisions.

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