scholarly journals Comprehensive synthetic genetic array analysis of alleles that interact with mutation of the Saccharomyces cerevisiae RecQ helicases Hrq1 and Sgs1

2020 ◽  
Author(s):  
Elsbeth Sanders ◽  
Phoebe A. Nguyen ◽  
Cody M. Rogers ◽  
Matthew L. Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Gene ◽  
Colony Growth ◽  
Strand Break ◽  
Telomere Maintenance ◽  
Temperature Sensitive ◽  
Recq Helicase ◽  
Synthetic Genetic Array ◽  
Sensitive Allele ◽  
Dna Maintenance

ABSTRACTMost eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgsl-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

scholarly journals Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1

2020 ◽  
Vol 10 (12) ◽  
pp. 4359-4368
Author(s):  
Elsbeth Sanders ◽  
Phoebe A. Nguyen ◽  
Cody M. Rogers ◽  
Matthew L. Bochman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Gene ◽  
Colony Growth ◽  
Strand Break ◽  
Telomere Maintenance ◽  
Temperature Sensitive ◽  
Recq Helicase ◽  
Synthetic Genetic Array ◽  
Link Type ◽  
Sensitive Allele

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

scholarly journals Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
Author(s):  
Xuan Wang ◽  
Grzegorz Ira ◽  
José Antonio Tercero ◽  
Allyson M. Holmes ◽  
John F. X. Diffley ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Temperature Sensitive ◽  
Replication Proteins ◽  
Dna Ligases ◽  
Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1389-1400
Author(s):  
Xiao Ming Zuo ◽  
G Desmond Clark-Walker ◽  
Xin Jie Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Nucleoids ◽  
Mtdna Replication ◽  
Sensitive Allele ◽  
Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae

1981 ◽  
Vol 1 (5) ◽  
pp. 460-468
Author(s):  
T Mizunaga ◽  
J S Tkacz ◽  
L Rodriguez ◽  
R A Hackel ◽  
J O Lampen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Parent Strain ◽  
Specific Activity ◽  
Single Gene ◽  
Companion Paper ◽  
Cellular Level ◽  
Temperature Sensitive ◽  
Mutagenic Treatment ◽  
Intracellular Degradation ◽  
Subunit Molecular Weight

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.

scholarly journals A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (4) ◽  
pp. 1879-1892 ◽  
Author(s):  
J L Davis ◽  
R Kunisawa ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Essential Gene ◽  
Single Gene ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sequence Element ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
Identical Manner

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

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 Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

2020 ◽  
Vol 98 (5) ◽  
pp. 624-630 ◽  
Author(s):  
Yanrui Zhu ◽  
Matthew D. Berg ◽  
Phoebe Yang ◽  
Raphaël Loll-Krippleber ◽  
Grant W. Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Genetic Disease ◽  
Elevated Temperatures ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Standard Genetic Code ◽  
Gene Encoding ◽  
Chromatid Cohesion ◽  
Sensitive Allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

scholarly journals Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

2020 ◽  
Author(s):  
Yanrui Zhu ◽  
Matthew D. Berg ◽  
Phoebe Yang ◽  
Raphaël Loll-Krippleber ◽  
Grant W. Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Amino Acid ◽  
Genetic Disease ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Standard Genetic Code ◽  
Gene Encoding ◽  
Chromatid Cohesion ◽  
Sensitive Allele

ABSTRACTMistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored function of the enzyme at elevated temperature. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

scholarly journals The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish

Development ◽  
2021 ◽  
Author(s):  
Tzu-Lun Tseng ◽  
Ying-Ting Wang ◽  
Chang-Yu Tsao ◽  
Yi-Teng Ke ◽  
Yi-Ching Lee ◽  
...  
Keyword(s):  
Positional Cloning ◽  
Single Gene ◽  
Rna Helicase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Juvenile Growth ◽  
Peter Pan ◽  
Organism Growth ◽  
Juvenile Stages ◽  
Sensitive Allele

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-Dependent RNA Helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

scholarly journals Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae.

Genetics ◽  
1995 ◽  
Vol 139 (4) ◽  
pp. 1521-1532 ◽  
Author(s):  
K Johzuka ◽  
H Ogawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Vegetative Growth ◽  
Strand Break ◽  
Double Strand Break ◽  
Heptad Repeat ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
C Terminus

Abstract A temperature-sensitive mre11-1 mutation of Saccharomyces cerevisiae causes defects in meiotic recombination and DNA repair during vegetative growth at a restrictive temperature. We cloned the MRE11 gene and found that it encodes a 643-amino acid protein with a highly acidic region containing a heptad repeat of Asp at its C-terminus and is located downstream of YMR44 near the RNA1 locus on the right arm of chromosome XIII. Transcripts of the MRE11 gene increased transiently and showed the same kinetics as that of the RAD50 gene during meiosis. In a mre11 disruption mutant (mre11::hisG), meiosis-specific double-strand break (DSB) formation is abolished. A comparison of the properties of mre11::hisG and a rad50 deletion mutant (rad50 delta) indicated that both mutants exhibited similar phenotypes in both meiosis and mitosis. Characterization of two double mutants, mre11::hisG rad50 delta and mre11::hisG rad50S, showed that MRE11 and RAD50 belong to the same epistasis group with respect to meiotic DSB formation and mitotic DNA repair. Using a two-hybrid system, we found that Mre11 interacts with Rad50 and itself in vivo. These results suggest that Mre11 and Rad50 proteins work in a complex in DSB formation and DNA repair during vegetative growth.

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