scholarly journals Mec1 and Rad53 Inhibit Formation of Single-Stranded DNA at Telomeres of Saccharomyces cerevisiae cdc13-1 Mutants

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 753-764 ◽  
Author(s):  
Xindan Jia ◽  
Ted Weinert ◽  
David Lydall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Minor Role ◽  
Clamp Loader ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Sliding Clamp ◽  
Telomere Binding Protein ◽  
A Minor ◽  
Dependent Pathway

Abstract Here we examine the roles of budding-yeast checkpoint proteins in regulating degradation of dsDNA to ssDNA at unprotected telomeres (in Cdc13 telomere-binding protein defective strains). We find that Rad17, Mec3, as well as Rad24, members of the putative checkpoint clamp loader (Rad24) and sliding clamp (Rad17, Mec3) complexes, are important for promoting degradation of dsDNA in and near telomere repeats. We find that Mec1, Rad53, as well as Rad9, have the opposite role: they inhibit degradation. Downstream checkpoint kinases Chk1 and Dun1 play no detectable role in either promoting degradation or inhibiting it. These data suggest, first, that the checkpoint sliding clamp regulates and/or recruits some nucleases for degradation, and, second, that Mec1 activates Rad9 to activate Rad53 to inhibit degradation. Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways. Exo1 appears to be targeted by the Mec1/Rad53-dependent pathway. Finally, analysis of double mutants suggests a minor role for Mec1 in promoting Rad24-dependent degradation of dsDNA. Thus, checkpoint proteins orchestrate carefully ssDNA production at unprotected telomeres.

scholarly journals Shugoshin Promotes Sister Kinetochore Biorientation in Saccharomyces cerevisiae

2008 ◽  
Vol 19 (3) ◽  
pp. 1199-1209 ◽  
Author(s):  
Brendan M. Kiburz ◽  
Angelika Amon ◽  
Adele L. Marston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Minor Role ◽  
Meiotic Cell ◽  
Homologous Chromosomes ◽  
Cell Divisions ◽  
Sister Chromatids ◽  
Sister Kinetochore ◽  
A Minor ◽  
Meiosis I

Chromosome segregation must be executed accurately during both mitotic and meiotic cell divisions. Sgo1 plays a key role in ensuring faithful chromosome segregation in at least two ways. During meiosis this protein regulates the removal of cohesins, the proteins that hold sister chromatids together, from chromosomes. During mitosis, Sgo1 is required for sensing the absence of tension caused by sister kinetochores not being attached to microtubules emanating from opposite poles. Here we describe a differential requirement for Sgo1 in the segregation of homologous chromosomes and sister chromatids. Sgo1 plays only a minor role in segregating homologous chromosomes at meiosis I. In contrast, Sgo1 is important to bias sister kinetochores toward biorientation. We suggest that Sgo1 acts at sister kinetochores to promote their biorientation.

scholarly journals Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Atasi Poddar ◽  
P. Todd Stukenberg ◽  
Daniel J. Burke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Spindle Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Checkpoint Proteins ◽  
In Cells

ABSTRACT Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

Nuclear behaviour and cell wall composition in relation to budding in mutants of the yeast Saccharomyces cerevisiae

1986 ◽  
Vol 64 (1) ◽  
pp. 193-200 ◽  
Author(s):  
Mario Lachapelle ◽  
E. Roger Boothroyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Spindle Pole ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Bud Emergence ◽  
Spindle Elongation ◽  
10 Nm Filaments ◽  
A Minor

A temperature-sensitive, cell division cycle mutant (cdc24–1) and karyogamy-deficient (kar1) mutant of Saccharomyces cerevisiae, both of which can produce binucleate or multinucleate cells, were used to study certain aspects of budding, after fluorescent staining for mannan, chitin, and nuclei (DNA). In most binucleate cells the two nuclei lay close together and divided into the same bud. In a few, however, the nuclei were far apart and one or two buds were formed, each proximal to a nucleus. The proximity of daughter nuclei in most blocked cdc24–1 cells suggests a role for the CDC24 gene product in spindle elongation. The relationship between the nuclei and the number and location of buds supports the theory of a preponderant role for the nucleus in budding. Although buds develop preferentially in regions of low chitin content in kar1 heterokaryons, the ability of cdc24–1 cells to bud even with a uniformly high content of chitin and mannan suggests a minor role for these cell wall constituents in determining the sites of bud emergence. The chitin ring is not needed for bud emergence but seems to play a role in normal bud development and in septum formation. Electron microscopy of cdc24–1 cells blocked (37 °C) for 8 h and released (23 °C) for 30 min showed morphologically normal spindle pole bodies, cytoplasmic microtubules, and intranuclear spindles. Although the chitin ring was absent, the ring of 10-nm filaments was present, consistent with its proposed role in bud emergence.

scholarly journals C-terminal HA tags compromise function and exacerbate phenotypes of Saccharomyces cerevisiae Bloom’s helicase homolog Sgs1 SUMOylation-associated mutants

2020 ◽  
Author(s):  
Matan Cohen ◽  
Michael Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Mitotic Cell ◽  
Minor Role ◽  
Mitotic Cell Cycle ◽  
Human Influenza ◽  
Wild Type ◽  
Epitope Tag ◽  
Influenza Hemagglutinin ◽  
A Minor

AbstractThe Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

scholarly journals A pathway of targeted autophagy is induced by DNA damage in budding yeast

2017 ◽  
Vol 114 (7) ◽  
pp. E1158-E1167 ◽  
Author(s):  
Vinay V. Eapen ◽  
David P. Waterman ◽  
Amélie Bernard ◽  
Nathan Schiffmann ◽  
Enrich Sayas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Budding Yeast ◽  
Strand Break ◽  
Central Component ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Damage Response ◽  
Autophagy Pathway ◽  
Yeast Mutants

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

scholarly journals Genetic Analysis of the TOR Pathway in Aspergillus nidulans

2005 ◽  
Vol 4 (9) ◽  
pp. 1595-1598 ◽  
Author(s):  
Gregory J. Fitzgibbon ◽  
Igor Y. Morozov ◽  
Meriel G. Jones ◽  
Mark X. Caddick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Aspergillus Nidulans ◽  
Minor Role ◽  
Tor Signaling ◽  
Tor Pathway ◽  
Genes Encoding ◽  
Tor Kinase ◽  
Mutant Phenotypes ◽  
A Minor

ABSTRACT We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

scholarly journals Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

2006 ◽  
Vol 175 (5) ◽  
pp. 729-741 ◽  
Author(s):  
Jorrit M. Enserink ◽  
Marcus B. Smolka ◽  
Huilin Zhou ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Morphology ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

Studies on Aldosterone Responsiveness to Angiotensin II during Clinical Variations in Calcium Metabolism in Normal Man

1982 ◽  
Vol 63 (3) ◽  
pp. 325-328 ◽  
Author(s):  
M. G. Bianchetti ◽  
C. Beretta-Piccoli ◽  
P. Weidmann ◽  
K. Boehringer ◽  
L. Link ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Calcium Metabolism ◽  
Normal Subjects ◽  
Plasma Aldosterone ◽  
Minor Role ◽  
Dose Rates ◽  
Normal Man ◽  
A Minor ◽  
Clinical Conditions ◽  
Dependent Pathway

1. Angiotensin II was infused at stepwise increasing dose rates (2, 4 and 10 pmol min–1 kg–1) in 12 normal subjects. Infusions were performed in the presence of normocalcaemia, mild hypercalcemia induced by concomitant calcium gluconate infusion, and after 2 weeks of treatment with nifedipine. 2. Pre-infusion plasma levels of angiotensin II, renin or aldosterone were not altered by acute mild hypercalcaemia or administration of nifedipine. The angiotensin II-induced increases in plasma aldosterone were also similar under the three study conditions. 3. Variations in calcium metabolism occurring under clinical conditions appear to play a minor role in modulating the angiotensin II-dependent pathway of aldosterone regulation in normal man.

scholarly journals Decreased Meiotic Intergenic Recombination and Increased Meiosis I Nondisjunction in exo1 Mutants of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (4) ◽  
pp. 1549-1557 ◽  
Author(s):  
David T Kirkpatrick ◽  
John R Ferguson ◽  
Thomas D Petes ◽  
Lorraine S Symington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homozygous Deletion ◽  
Minor Role ◽  
Strand Breaks ◽  
Exonuclease I ◽  
Primary Activity ◽  
Dna Mismatches ◽  
Intergenic Recombination ◽  
A Minor ◽  
Meiosis I

Abstract Exonuclease I was originally identified as a 5′ → 3′ deoxyribonuclease present in fractionated extracts of Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genetic analysis of exo1 mutants of both yeasts revealed no major defect in meiosis, suggesting that exonuclease I is unlikely to be the primary activity that processes meiosis-specific double-strand breaks (DSBs). We report here that exo1 mutants of S. cerevisiae exhibit subtle but complex defects in meiosis. Diploids containing a homozygous deletion of EXO1 show decreased spore viability associated with an increase in meiosis I nondisjunction, while intergenic recombination is reduced about twofold. Exo1p functions in the same pathway as Msh5p for intergenic recombination. The length of heteroduplex tracts within the HIS4 gene is unaffected by the exo1 mutation. These results suggest that Exo1p is unlikely to play a major role in processing DSBs to form single-stranded tails at HIS4, but instead appears to promote crossing over to ensure disjunction of homologous chromosomes. In addition, our data indicate that exonuclease I may have a minor role in the correction of large DNA mismatches that occur in heteroduplex DNA during meiotic recombination at the HIS4 locus.

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