TEN1 Is Essential for CDC13-Mediated Telomere Capping

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

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

Genetics ◽

10.1093/genetics/153.2.595 ◽

1999 ◽

Vol 153 (2) ◽

pp. 595-605 ◽

Cited By ~ 2

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.

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New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

MGG Molecular & General Genetics ◽

10.1007/bf00384381 ◽

1982 ◽

Vol 187 (1) ◽

pp. 42-46 ◽

Cited By ~ 35

Author(s):

Lawrence B. Dumas ◽

Joan P. Lussky ◽

Elizabeth J. McFarland ◽

Janis Shampay

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants

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GSP1 and GSP2, genetic suppressors of the prp20-1 mutant in Saccharomyces cerevisiae: GTP-binding proteins involved in the maintenance of nuclear organization.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2152 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2152-2161 ◽

Cited By ~ 83

Author(s):

P Belhumeur ◽

A Lee ◽

R Tam ◽

T DiPaolo ◽

N Fortin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Essential Gene ◽

Negative Control ◽

Temperature Sensitive ◽

Vitro Assay ◽

Gtp Binding ◽

Genetic Suppressors ◽

Multicopy Suppressors

The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.

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Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽

10.1093/genetics/148.3.989 ◽

1998 ◽

Vol 148 (3) ◽

pp. 989-1005 ◽

Cited By ~ 5

Author(s):

Keiko Umezu ◽

Neal Sugawara ◽

Clark Chen ◽

James E Haber ◽

Richard D Kolodner

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Physical Analysis ◽

Single Stranded Dna ◽

Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1000 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1000-1012 ◽

Cited By ~ 24

Author(s):

M N Conrad ◽

C S Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Nuclear Genome ◽

Electron Microscopic Examination ◽

Temperature Sensitive ◽

Chromosomal Dna ◽

Electron Microscopic ◽

Dna Strands ◽

Total Dna ◽

Random Fraction

Chromosomal DNA replication was examined in temperature-sensitive mutants of Saccharomyces cerevisiae defective in a gene required for the completion of S phase at the nonpermissive temperature, 37 degrees C. Based on incorporation of radioactive precursors and density transfer experiments, strains carrying three different alleles of cdc2 failed to replicate approximately one-third of their nuclear genome at 37 degrees C. Whole-cell autoradiography experiments demonstrated that 93 to 96% of the cells synthesized DNA at 37 degrees C. Therefore, all cells failed to replicate part of their genome. DNA isolated from terminally arrested cells was of normal size as measured on neutral and alkaline sucrose gradients, suggesting that partially replicated DNA molecules do not accumulate and that DNA strands are ligated properly in cdc2 mutants. In addition, electron microscopic examination of the equivalent of more than one genome's DNA from arrested cells failed to reveal any partially replicated molecules. The sequences which failed to replicate at 37 degrees C were not highly specific; eight different cloned sequences replicated to the same extent as total DNA. The 2-microns plasmid DNA and rDNA replicated significantly less well than total DNA, but approximately one-half of these sequences replicated at 37 degrees C. These observations suggest that cdc2 mutants are defective in an aspect of initiation of DNA replication common to all chromosomes such that a random fraction of the chromosomes fail to initiate replication at 37 degrees C, but that once initiated, replication proceeds normally.

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Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3242 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3242-3253 ◽

Cited By ~ 68

Author(s):

Y Shu ◽

H Yang ◽

E Hallberg ◽

R Hallberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

High Temperatures ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Molecular Genetic Analysis ◽

Regulatory Subunit ◽

Temperature Sensitive ◽

Checkpoint Control ◽

Phosphatase 2A

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.

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DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.365-376.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 365-376

Author(s):

M E Budd ◽

K D Wittrup ◽

J E Bailey ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Polymerase ◽

Temperature Sensitive ◽

Dna Polymerase I ◽

X Ray ◽

Temperature Sensitive Mutants ◽

Polymerase I ◽

Dna Polymerase Ii

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

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The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression

Journal of Cell Science ◽

10.1242/jcs.113.7.1199 ◽

2000 ◽

Vol 113 (7) ◽

pp. 1199-1211

Author(s):

G. Buscemi ◽

F. Saracino ◽

D. Masnada ◽

M.L. Carbone

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Actin Cytoskeleton ◽

Cell Cycle Progression ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Cycle Progression ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

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Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation

Molecular and Cellular Biology ◽

10.1128/mcb.3.10.1730-1737.1983 ◽

1983 ◽

Vol 3 (10) ◽

pp. 1730-1737

Author(s):

C L Kuo ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Recombinant Plasmid ◽

Yeast Genome ◽

Temperature Sensitive ◽

Deletion Mapping ◽

Nonpermissive Temperature ◽

Recombinant Plasmids ◽

Selection For ◽

Cloned Gene

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp50 bearing the yeast ARS1, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product.

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Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences

10.1101/2020.05.29.123083 ◽

2020 ◽

Author(s):

Thareendra De Zoysa ◽

Eric M. Phizicky

Keyword(s):

Saccharomyces Cerevisiae ◽

Quality Control ◽

Amino Acid ◽

Temperature Sensitivity ◽

Temperature Sensitive ◽

Temperature Resistance ◽

General Amino Acid Control ◽

Growth Defects ◽

Acid Control ◽

Body Modifications

AbstractAll tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5’-3’ exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 of tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the temperature sensitivity of trm8Δ mutants was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA decay and enhanced temperature sensitivity. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.Author SummarytRNA modifications are highly conserved and their lack frequently results in growth defects in the yeast Saccharomyces cerevisiae and neuorological disorders in humans. S. cerevsiaie has two tRNA quality control decay pathways that sense tRNAs lacking modifications in the main tRNA body. One of these, the rapid tRNA decay (RTD) pathway, targets mature tRNAs for 5’-3’ exonucleolytic decay by Rat1 and Xrn1. It is unknown if RTD is conserved in eukaryotes, and if it might explain phenotypes associated with body modification defects. Here we focus on trm8Δ mutants, lacking m7G46, in the evolutionarily distant yeast Schizosaccharomyces pombe. Loss of m7G causes temperature sensitivity and RTD in S. cerevisiae, microcephalic primordial dwarfism in humans, and defective stem cell renewal in mice. We show that S. pombe trm8Δ mutants are temperature sensitive due to tY(GUA) decay by Rat1, implying conservation of RTD among divergent eukaryotes. We also show that the onset of RTD triggers activation of the general amino acid control (GAAC) pathway in both S. pombe and S. cerevisiae, resulting in exacerbated decay in S. pombe and reduced decay in S. cerevisiae. We speculate that RTD and its regulation of the GAAC pathway will be widely conserved in eukaryotes including humans.

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