Details and Concerns Regarding the G2/M DNA Damage Checkpoint in Budding Yeast

Weinert, Ted; Little, E.J.; Shanks, Lisa; Admire, Anthony A.; Gardner, Richard D.; Putnam, Charles W.; Michelson, Rhett J.; Nyberg, Kara A.; Sundareshan, Padma

doi:10.1101/sqb.2000.65.433

Cold Spring Harbor Symposia on Quantitative Biology

2000

DOI: 10.1101/sqb.2000.65.433

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Details and Concerns Regarding the G2/M DNA Damage Checkpoint in Budding Yeast

Ted Weinert¹,

E.J. Little²,

Lisa Shanks³

et al.

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Cited by 3 publications

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“…Chk1 has a well-described role in promoting cell cycle arrest by phosphorylating and thereby stabilizing Pds1 (the Saccharomyces cerevisiae securin) (17)(18)(19). Rad53 phosphorylates many downstream substrates, resulting in a block to late-origin firing, cell cycle arrest, and activation of the Dun1 kinase (20)(21)(22)(23)(24). Although prior work has examined the mechanism by which the DNA damage checkpoint promotes transcriptional induction of its target genes, the mechanisms of transcriptional inhibition are less well understood (25).…”

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Rad53 Downregulates Mitotic Gene Transcription by Inhibiting the Transcriptional Activator Ndd1

Edenberg

Vashisht

Benanti

et al. 2014

Molecular and Cellular Biology

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The 33 genes in the Saccharomyces cerevisiae mitotic CLB2 transcription cluster have been known to be downregulated by the DNA damage checkpoint for many years. Here, we show that this is mediated by the checkpoint kinase Rad53 and the dedicated transcriptional activator of the cluster, Ndd1. Ndd1 is phosphorylated in response to DNA damage, which blocks recruitment to promoters and leads to the transcriptional downregulation of the CLB2 cluster. Finally, we show that downregulation of Ndd1 is an essential function of Rad53, as a hypomorphic ndd1 allele rescues RAD53 deletion. Over a decade ago, a group of mitotic genes called the CLB2 cluster was shown to be transcriptionally downregulated in response to DNA damage (1). The CLB2 cluster consists of 33 coregulated mitotic genes, including those for its namesake, Clb2 (a B-type cyclin), Cdc5 (Polo kinase), Cdc20 (the activator of the anaphase promoting complex), Hst3 (a sirtuin histone deacetylase), and many others (3). Because so many important mitotic regulators are part of this cluster, it is a hub for the regulation of mitosis during the cell cycle and in response to DNA damage.In an unperturbed cell cycle, CLB2 cluster transcription is tightly regulated, with transcription off during G 1 phase and high in early mitosis (3-5). Throughout the cell cycle, Mcm1 and Fkh2 are present at the promoters of these mitotic genes and coordinate both repression and activation by recruiting additional transcriptional regulators (4, 5). During G 1 , transcription of this cluster is off. As cells enter S phase, Clb5-cyclin-dependent kinase (CDK) phosphorylates Fkh2 (6). Later, Clb2-CDK and Cdc5 phosphorylate Ndd1, which leads to Ndd1's recruitment to its target promoters through an interaction with Fkh2 (7,8,27). Ndd1 then functions as a transcriptional activator to drive high levels of CLB2 cluster transcription (4,8). Ndd1 is itself cell cycle regulated and is transcribed in early S phase (9, 10). As both CLB2 and CDC5 are CLB2 cluster members themselves, these phosphorylations generate a positive-feedback loop that drives switch-like transcription of the cluster. In addition, the precise timing of transcription of this cluster is modulated by protein kinase C (PKC) (11) and, for a subset of members, Yox1 (12, 13).In response to DNA damage agents, transcription of the CLB2 cluster is downregulated by the DNA damage checkpoint (1, 2). This checkpoint is a signal transduction cascade that is activated in response to genotoxic stress, and the checkpoint is required to prevent replication fork collapse and arrest the cell cycle (14, 15). At the top of the kinase cascade that makes up the checkpoint, the phosphatidylinositide 3-kinase-like kinase Mec1 (the homolog of human ATR) is activated. Mec1 then phosphorylates and activates downstream effector kinases Chk1 and Rad53 (homologs of human Chk1 and Chk2, respectively) (16). Chk1 has a well-described role in promoting cell cycle arrest by phosphorylating and thereby stabilizing Pds1 (the Saccharomyces cerevisiae securin) (17-19...

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mentioning

confidence: 99%

Rad53 Downregulates Mitotic Gene Transcription by Inhibiting the Transcriptional Activator Ndd1

Edenberg

Vashisht

Benanti

et al. 2014

Molecular and Cellular Biology

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Current Awareness

2001

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals ‐ search completed 8th. Aug. 2001)

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Abrogation of the Chk1-Pds1 Checkpoint Leads to Tolerance of Persistent Single-Strand Breaks in Saccharomyces cerevisiae

Karumbati

Wilson

2005

Genetics

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In budding yeast, Apn1, Apn2, Tpp1, and Rad1/Rad10 are important enzymes in the removal of spontaneous DNA lesions. apn1 apn2 rad1 yeast are inviable due to accumulation of abasic sites and strand breaks with 3Ј blocking lesions. We found that tpp1 apn1 rad1 yeast exhibited slow growth but frequently gave rise to spontaneous slow growth suppressors that segregated as single-gene mutations. Using a candidate gene approach, we identified several tpp1 apn1 rad1 suppressors. Deleting uracil glycosylase suppressed both tpp1 apn1 rad1 and apn1 apn2 rad1 growth defects by reducing the abasic site burden. Mutants affecting the Chk1-Pds1 metaphase-anaphase checkpoint only suppressed tpp1 apn1 rad1 slow growth. In contrast, most S-phase checkpoint mutants were synthetically lethal in a tpp1 apn1 rad1 background. Epistasis analyses showed an additive effect between chk1 and ung1, indicating different mechanisms of suppression. Loss of Chk1 partially restored cell-growth parameters in tpp1 apn1 rad1 yeast, but at the same time exacerbated chromosome instability. We propose a model in which recombinational repair during S phase coupled with failure of the metaphase-anaphase checkpoint allows for tolerance of persistent single-strand breaks at the expense of genome stability. ous lesion leading to toxicity in these strains.gene replacement was also confirmed by the loss of product in PCR using primers in the coding region of the targeted gene.We have found that a haploid strain lacking Apn1, et al. 1994Sanchez et al. 1999). There is the samples were then measured at 15-to 30-min intervals by using a GENESYS20 spectrophotometer for at least two considerable overlap in the proteins required for these from at least six different isolates were analyzed. As with theIn this study we found that tpp1 apn1 rad1 mutant tetrad scores, this was done prior to genotyping, so that all yeast spontaneously gave rise to single-gene slow-growth data were recorded blind to the strain genotype.suppressors at a high frequency. Using a candidate gene Plasmid loss assay: Plasmid loss rates were determined using the method described by Huang and Koshland (2003).approach, we observed that disruption of the M-A checkBriefly, YW1495, a diploid strain carrying the pRS412 plasmid point conferred suppression of tpp1 apn1 rad1 slow with the CEN/ARS and the ADE2 marker gene, was dissected growth. In contrast, deletion of S-phase checkpoint on selective media plates to get the strains with appropriate genes either had no effect or conferred synthetic lethalgenotypes and containing the plasmid. Either a 6-day tetrad ity in a tpp1 apn1 rad1 mutant background. The implicacolony or a wild-type strain (YW465) carrying the plasmid was used to inoculate the initial culture and allowed to grow in tions of these findings are discussed.YPAD for approximately six to seven doublings. In the case of slow-growing strains, three or more colonies were pooled to form the initial inoculum. The plasmid loss rate was calculated MATERIALS AND METHODS using the formula: l...

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Details and Concerns Regarding the G2/M DNA Damage Checkpoint in Budding Yeast

Cited by 3 publications

References 53 publications

Rad53 Downregulates Mitotic Gene Transcription by Inhibiting the Transcriptional Activator Ndd1

Rad53 Downregulates Mitotic Gene Transcription by Inhibiting the Transcriptional Activator Ndd1

Current Awareness

Abrogation of the Chk1-Pds1 Checkpoint Leads to Tolerance of Persistent Single-Strand Breaks in Saccharomyces cerevisiae

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