scholarly journals The Saccharomyces cerevisiae DNA Recombination and Repair Functions of the RAD52 Epistasis Group Inhibit Ty1 Transposition

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 543-556 ◽  
Author(s):  
Alison J Rattray ◽  
Brenda K Shafer ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Dna Recombination ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Low Frequencies ◽  
Double Stranded Dna ◽  
High Level ◽  
In Cells

Abstract RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or δ) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

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 The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway.

Genetics ◽  
1990 ◽  
Vol 124 (4) ◽  
pp. 817-831 ◽  
Author(s):  
R H Schiestl ◽  
S Prakash ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Gamma Ray ◽  
Dna Lesions ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Uv Mutagenesis ◽  
Uv Sensitivity ◽  
Suppressor Mutations ◽  
Induced Mutagenesis

Abstract rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, we have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6 delta) mutations and show that they also suppress the gamma-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of gamma-ray sensitivity. The six suppressor mutations we isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. We show that suppression of rad6 delta is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6 delta SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed.

Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway

1990 ◽  
Vol 10 (2) ◽  
pp. 510-517
Author(s):  
G M Cole ◽  
D E Stone ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mating Pheromone ◽  
Gal1 Promoter ◽  
Gamma Subunits ◽  
Adaptation Response ◽  
High Level

The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.

scholarly journals Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (12) ◽  
pp. 6229-6247 ◽  
Author(s):  
S M Miller ◽  
B Magasanik
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Glutamate Dehydrogenase ◽  
Regulatory System ◽  
Sole Source ◽  
Lacz Fusion ◽  
Upstream Region ◽  
In Cells

We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

scholarly journals Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

2001 ◽  
Vol 21 (6) ◽  
pp. 2048-2056 ◽  
Author(s):  
Laurence Signon ◽  
Anna Malkova ◽  
Maria L. Naylor ◽  
Hannah Klein ◽  
James E. Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Telomere Maintenance ◽  
Diploid Cells ◽  
Break Induced Replication ◽  
In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

scholarly journals Helicase Mechanisms During Homologous Recombination inSaccharomyces cerevisiae

2019 ◽  
Vol 48 (1) ◽  
pp. 255-273 ◽  
Author(s):  
J. Brooks Crickard ◽  
Eric C. Greene
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleic Acid ◽  
Homologous Recombination ◽  
Nucleic Acids ◽  
Acid Metabolism ◽  
Model Organism ◽  
Nucleic Acid Metabolism ◽  
Repair Pathway ◽  
Unidirectional Movement ◽  
Nucleoprotein Complexes

Helicases are enzymes that move, manage, and manipulate nucleic acids. They can be subdivided into six super families and are required for all aspects of nucleic acid metabolism. In general, all helicases function by converting the chemical energy stored in the bond between the gamma and beta phosphates of adenosine triphosphate into mechanical work, which results in the unidirectional movement of the helicase protein along one strand of a nucleic acid. The results of this translocation activity can range from separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. In this review, we focus on describing key helicases from the model organism Saccharomyces cerevisiae that contribute to the regulation of homologous recombination, which is an essential DNA repair pathway for fixing damaged chromosomes.

Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1418-1418
Author(s):  
Masood A. Shammas ◽  
Hemant Koley ◽  
Sima Shah ◽  
Ramesh B. Batchu ◽  
Pierfrancesco Tassone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Cell Lines ◽  
Plasma Cells ◽  
Dna Recombination ◽  
Endonuclease Activity ◽  
Single Nucleotide ◽  
Genetic Changes

Abstract Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.

Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation

2005 ◽  
Vol 33 (6) ◽  
pp. 1447-1450 ◽  
Author(s):  
M.A. Macris ◽  
P. Sung
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Dna Replication ◽  
Homologous Recombination ◽  
High Energy ◽  
Yeast Saccharomyces Cerevisiae ◽  
Major Pathway ◽  
Dna Dsbs ◽  
Srs2 Helicase

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic analyses in the yeast Saccharomyces cerevisiae have revealed a central role of the Srs2 helicase in preventing untimely HR events and in inhibiting the formation of potentially deleterious DNA structures or nucleoprotein complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2 in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in order to preserve genomic integrity and promote cell survival.

scholarly journals Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (7) ◽  
pp. 2329-2334 ◽  
Author(s):  
J R Simon ◽  
P D Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Mutant Gene ◽  
Transformation Process ◽  
Single Stranded Dna ◽  
Autonomous Replication ◽  
Double Stranded Dna ◽  
Chromosomal Genes ◽  
Chromosomal Sequences

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

scholarly journals Saccharomyces cerevisiae histone Chaperones FACT and Spt6 modulate Pol II and histone occupancy genome-wide

2018 ◽  
Author(s):  
Rakesh Pathak ◽  
Priyanka Singh ◽  
Sudha Ananthakrishnan ◽  
Sarah Adamczyk ◽  
Olivia Schimmel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Histone Chaperones ◽  
Strongly Correlated ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Coding Regions ◽  
Genome Wide ◽  
High Level

ABSTRACTHistone chaperones, chromatin remodelers, and histone modifying complexes play a critical role in alleviating the nucleosomal barrier. Here, we have examined the role of two highly conserved yeast (Saccharomyces cerevisiae) histone chaperones, FACT and Spt6, in regulating transcription and histone occupancy. We show that the H3 tail contributes to the recruitment of FACT to coding sequences in a manner dependent on acetylation. We found that deleting a H3 HAT Gcn5 or mutating lysines on the H3 tail impairs FACT recruitment at ADH1 and ARG1 genes. However, deleting the H4 tail or mutating the H4 lysines failed to dampen FACT occupancy in coding regions. Additionally, we show that FACT-depletion greatly reduces Pol II occupancy in the 5’ ends genome-wide. By contrast, Spt6-depletion led to reduction in Pol II occupancy towards the 3’ end, in a manner dependent on the gene-length. Severe transcription and histone eviction defects were also observed in a strain that was impaired for Spt6 recruitment (spt6Δ202) and depleted of FACT. Importantly, the severity of the defect strongly correlated with WT Pol II occupancies at these genes, indicating critical roles of Spt6 and Spt16 in promoting high-level transcription. Collectively, our study shows cooperation, as well as redundancy between chaperones, FACT and Spt6, in regulating transcription and chromatin in coding regions of transcribed genes.

Sign in / Sign up

Export Citation Format

Share Document