scholarly journals MEC3, MEC1, and DDC2Are Essential Components of a Telomere Checkpoint Pathway Required for Cell Cycle Arrest during Senescence in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (8) ◽  
pp. 2626-2638 ◽  
Author(s):  
Shinichiro Enomoto ◽  
Lynn Glowczewski ◽  
Judith Berman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Telomerase Activity ◽  
Cellular Level ◽  
Dna Damage Checkpoint ◽  
Average Growth ◽  
Progressive Decline ◽  
Checkpoint Response ◽  
Essential Components

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, andDDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1,RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.

scholarly journals Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

2006 ◽  
Vol 26 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Guochun Jiang ◽  
Aziz Sancar
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Human Cells ◽  
Dna Damage Checkpoint ◽  
Damage Site ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Transcription Complexes

ABSTRACT We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

scholarly journals Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

2007 ◽  
Vol 27 (19) ◽  
pp. 6852-6862 ◽  
Author(s):  
Aimin Peng ◽  
Andrea L. Lewellyn ◽  
James L. Maller
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Nuclear Dna ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Cycle Checkpoint ◽  
Damaged Dna

ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

scholarly journals Loss of Rereplication Control in Saccharomyces cerevisiae Results in Extensive DNA Damage

2005 ◽  
Vol 16 (1) ◽  
pp. 421-432 ◽  
Author(s):  
Brian M. Green ◽  
Joachim J. Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Genome Stability ◽  
Eukaryotic Cell ◽  
Entire Genome ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Response ◽  
Checkpoint Protein ◽  
C Terminus

To maintain genome stability, the entire genome of a eukaryotic cell must be replicated once and only once per cell cycle. In many organisms, multiple overlapping mechanisms block rereplication, but the consequences of deregulating these mechanisms are poorly understood. Here, we show that disrupting these controls in the budding yeast Saccharomyces cerevisiae rapidly blocks cell proliferation. Rereplicating cells activate the classical DNA damage-induced checkpoint response, which depends on the BRCA1 C-terminus checkpoint protein Rad9. In contrast, Mrc1, a checkpoint protein required for recognition of replication stress, does not play a role in the response to rereplication. Strikingly, rereplicating cells accumulate subchromosomal DNA breakage products. These rapid and severe consequences suggest that even limited and sporadic rereplication could threaten the genome with significant damage. Hence, even subtle disruptions in the cell cycle regulation of DNA replication may predispose cells to the genomic instability associated with tumorigenesis.

scholarly journals Yeast Securin is trafficked through the endomembrane system to regulate cell cycle arrest during the DNA damage response

2019 ◽  
Author(s):  
Brenda R. Lemos ◽  
David P. Waterman ◽  
James E. Haber
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Ubiquitin Proteasome System ◽  
Dna Damage Checkpoint ◽  
Transport Pathway ◽  
Checkpoint Response ◽  
Cycle Arrest ◽  
Chromatid Segregation ◽  
Ubiquitin Proteasome

AbstractThe yeast securin protein, Pds1, belongs to a class of highly conserved eukaryotic proteins that regulate the timing of chromatid segregation during mitosis by inhibiting separase, Esp1. During the metaphase to anaphase transition Pds1 is degraded by the ubiquitin-proteasome system in the nucleus, unleashing Esp1’s protease activity to cleave cohesin surrounding sister chromatids. In response to DNA damage Pds1 is phosphorylated and stabilized, stalling mitotic progression to preserve genomic integrity. In addition, during the DNA damage checkpoint response, securin and separase are partially localized in the vacuole. Here we genetically dissect the requirements for securin’s vacuolar localization and find that it is dependent on the Alkaline Phosphatase (ALP) endosomal transport pathway but not autophagy. Blocking retrograde traffic between the Golgi and the endoplasmic reticulum, by inhibiting COPI vesicle transport, drives Pds1 into the vacuole, whereas inhibiting antegrade transport by disrupting COPII-mediated traffic, results in the unexpected loss of Pds1 and the extinction of DNA damage-induced cell cycle arrest. We report that the induction of ER stress, either genetically or by treating cells with dithiothreitol, is sufficient to extinguish the DNA damage checkpoint, suggesting crosstalk between these two pathways. These data highlight new ways in which Pds1 and Esp1 are regulated during a DNA damage induced G2/M arrest and its requirement in the maintenance of the DNA damage checkpoint response.

Regulation of Septum Formation in Aspergillus nidulans by a DNA Damage Checkpoint Pathway

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 1055-1067
Author(s):  
Steven D Harris ◽  
Peter R Kraus
Keyword(s):  
Dna Damage ◽  
Aspergillus Nidulans ◽  
Cellular Response ◽  
Dna Damage Checkpoint ◽  
Temperature Sensitive ◽  
Dna Metabolism ◽  
Chromosomal Dna ◽  
Checkpoint Response ◽  
Septum Formation ◽  
Checkpoint Pathway

Abstract In Aspergillus nidulans, germinating conidia undergo multiple rounds of nuclear division before the formation of the first septum. Previous characterization of temperature-sensitive sepB and sepJ mutations showed that although they block septation, they also cause moderate defects in chromosomal DNA metabolism. Results presented here demonstrate that a variety of other perturbations of chromosomal DNA metabolism also delay septum formation, suggesting that this is a general cellular response to the presence of sublethal DNA damage. Genetic evidence is provided that suggests that high levels of cyclin-dependent kinase (cdk) activity are required for septation in A. nidulans. Consistent with this notion, the inhibition of septum formation triggered by defects in chromosomal DNA metabolism depends upon Tyr-15 phosphorylation of the mitotic cdk p34nimX. Moreover, this response also requires elements of the DNA damage checkpoint pathway. A model is proposed that suggests that the DNA damage checkpoint response represents one of multiple sensory inputs that modulates p34nimX activity to control the timing of septum formation.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

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