Upregulation of dNTP Levels After Telomerase Inactivation Influences Telomerase-Independent Telomere Maintenance Pathway Choice in Saccharomyces cerevisiae

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.

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Global Phenotype Screening and Transcript Analysis Outlines the Inhibitory Mode(s) of Action of Two Amphibian-Derived, α-Helical, Cationic Peptides on Saccharomyces cerevisiae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01007-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 3948-3959 ◽

Cited By ~ 25

Author(s):

C. Oliver Morton ◽

Andrew Hayes ◽

Michael Wilson ◽

Bharat M. Rash ◽

Stephen G. Oliver ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Inhibitory Action ◽

Protein Targeting ◽

Telomere Maintenance ◽

Yeast Cells ◽

Membrane Disruption ◽

Dna Integrity ◽

Cationic Peptides

ABSTRACT Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an α-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae. Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.

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Control of Translocations between Highly Diverged Genes by Sgs1, the Saccharomyces cerevisiae Homolog of the Bloom'sSyndrome Protein

Molecular and Cellular Biology ◽

10.1128/mcb.00161-06 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5406-5420 ◽

Cited By ~ 52

Author(s):

Kristina H. Schmidt ◽

Joann Wu ◽

Richard D. Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Genome Stability ◽

Dna Helicase ◽

Telomere Maintenance ◽

Template Switching ◽

Checkpoint Kinase ◽

Genes Encoding ◽

Atm Protein ◽

Break Induced Replication

ABSTRACT Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching.

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Role of folding kinetics of secondary structures in telomeric G-overhangs in the regulation of telomere maintenance in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012914 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8958-8971 ◽

Cited By ~ 3

Author(s):

Katarina Jurikova ◽

Martin Gajarsky ◽

Mona Hajikazemi ◽

Jozef Nosek ◽

Katarina Prochazkova ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Secondary Structures ◽

Telomere Maintenance ◽

Folding Kinetics ◽

Mobility Shift ◽

Dna Structures ◽

Electrophoretic Mobility Shift Assays ◽

Kinetics Of

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

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Ty1 Mobilizes Subtelomeric Y′ Elements in Telomerase-Negative Saccharomyces cerevisiae Survivors

Molecular and Cellular Biology ◽

10.1128/mcb.24.22.9887-9898.2004 ◽

2004 ◽

Vol 24 (22) ◽

pp. 9887-9898 ◽

Cited By ~ 30

Author(s):

Patrick H. Maxwell ◽

Candice Coombes ◽

Alison E. Kenny ◽

Joseph F. Lawler ◽

Jef D. Boeke ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Cell Division ◽

Long Terminal Repeat ◽

Reverse Transcription ◽

Telomere Maintenance ◽

Nonpermissive Temperature ◽

Telomeric Dna ◽

Copy Numbers ◽

Ty1 Retrotransposition

ABSTRACT When telomerase is inactivated in Saccharomyces cerevisiae, telomeric DNA shortens with every cell division, and cells stop dividing after ∼100 generations. Survivors that form in these senescent populations and resume growing have variably amplified arrays of subtelomeric Y′ elements. We marked a chromosomal Y′ element with the his3AI retrotransposition indicator gene and found that Y′HIS3 cDNA was incorporated into the genome at ∼10- to 1,000-fold-higher frequencies in survivors compared to telomerase-positive strains. Y′HIS3 cDNA mobility was significantly reduced if assayed at 30°C, a nonpermissive temperature for Ty1 retrotransposition, or in the absence of Tec1p, a transcription factor for Ty1. Microarray analysis revealed that Y′ RNA is preferentially associated with Ty1 virus-like particles (VLPs). Genomic copies of Y′HIS3 cDNA typically have downstream oligo(A) tracts, followed by a complete Ty1 long terminal repeat and TYA1 or TYB1 sequences. These data are consistent with the use of Ty1 cDNA to prime reverse transcription of polyadenylated Y′ RNA within Ty1 VLPs. Unmarked Y′-oligo(A)-Ty1 cDNA was also detected in survivors, reaching copy numbers of ∼10−2 per genome. We propose that Y′-oligo(A)-Ty1 cDNA recombines with Y′ elements at eroding telomeres in survivors and may play a role in telomere maintenance in the absence of telomerase.

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EXO1 Contributes to Telomere Maintenance in Both Telomerase-Proficient and Telomerase-Deficient Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.166.4.1651 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1651-1659 ◽

Cited By ~ 60

Author(s):

A. A. Bertuch

Keyword(s):

Saccharomyces Cerevisiae ◽

Telomere Maintenance

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Suppression of cdc13-2-associated senescence by pif1-m2 requires Ku-mediated telomerase recruitment

10.1101/2021.03.31.437837 ◽

2021 ◽

Author(s):

Enikő Fekete-Szücs ◽

Fernando Rodrigo Rosas Bringas ◽

Sonia Stinus ◽

Michael Chang

Keyword(s):

Saccharomyces Cerevisiae ◽

Replicative Senescence ◽

Telomere Maintenance ◽

Telomerase Rna ◽

Telomeric Dna ◽

Insight Into

In Saccharomyces cerevisiae, recruitment of telomerase to telomeres requires an interaction between Cdc13, which binds single-stranded telomeric DNA, and the Est1 subunit of telomerase. A second pathway involving an interaction between the yKu complex and telomerase RNA (TLC1) contributes to telomerase recruitment, but cannot sufficiently recruit telomerase on its own to prevent replicative senescence when the primary Cdc13-Est1 pathway is abolished—for example, in the cdc13-2 mutant. In this study, we find that mutation of PIF1, which encodes a helicase that inhibits telomerase, suppresses the replicative senescence of cdc13-2 by increasing reliance on the yKu-TLC1 pathway for telomerase recruitment. Our findings reveal new insight into telomerase-mediated telomere maintenance.

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

10.1101/2020.08.28.272666 ◽

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.

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