Survival of DNA Damage in Yeast Directly Depends on Increased dNTP Levels Allowed by Relaxed Feedback Inhibition of Ribonucleotide Reductase

Chabes, Andrei; Georgieva, Bilyana; Domkin, V. D.; Zhao, Xiaolan; Rothstein, Rodney; Thelander, Lars

doi:10.1016/s0092-8674(03)00075-8

Cited by 390 publications

(491 citation statements)

References 42 publications

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“…Mammalian cell response to DNA damage is somewhat ambiguous since neither logarithmically growing nor synchronized cells show accumulation of dNTP pools after DNA damage [12]. Our data are consistent with the idea that cells accumulate dNTPs in response to DNA damage as suggested by Chabes et al [11]. Although the DNA damage response and dNTP synthesis pathways of yeast and mammals have significant similarities, it is neither clear why mammalian cells do not show an increase in the sizes of their dNTP pools nor how they perform the repair process in the event of DNA damage.…”

Section: Discussionsupporting

confidence: 90%

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Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

Koç

Merrill

2007

Biochemical and Biophysical Research Communications

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Deoxyribonucleotide pools are maintained at levels that support efficient and yet accurate DNA replication and repair. Rad53 is part of a protein kinase regulatory cascade that, conceptually, promotes dNTP accumulation in four ways: (1) it activates the transcription of ribonucleotide reductase subunits by inhibiting the Crt1 repressor; (2) it plays a role in relocalization of ribonucleotide reductase subunits RNR2 and RNR4 from nucleus to cytoplasm; (3) it antagonizes the action of Sml1, a protein that binds and inhibits ribonucleotide reductase; and (4) it blocks cell-cycle progression in response to DNA damage, thus preventing dNTP consumption through replication forks. Although several lines of evidence support the above modes of Rad53 action, an effect of a rad53 mutation on dNTP levels has not been directly demonstrated. In fact, in a previous study, a rad53-11 mutation did not result in lower dNTP levels in asynchronous cells or in synchronized cells that entered the S-phase in the presence of the RNR inhibitor hydroxyurea. These anomalies prompted us to investigate whether the rad53-11 mutation affected dNTP levels in cells exposed to a DNA-damaging dose of ethylmethyl sulfonate (EMS). Although dNTP levels increased by 2-to 3-fold in EMS treated wild-type cells, rad53-11 cells showed no such change. Thus, the results indicate that Rad53 checkpoint function is not required for dNTP pool maintenance in normally growing cells, but is required for dNTP pool expansion in cells exposed to DNA-damaging agents. Ó 2006 Elsevier Inc. All rights reserved.Keywords: Rad53; dNTP; DNA damage; RNR; EMS; Yeast Cells have evolved mechanisms to maintain or augment deoxyribonucleotide triphosphate levels during DNA synthesis and repair. Yeast cells respond to DNA damage and replication block by arresting the progression of the cell cycle at specific points and by inducing the expression of genes thought to facilitate DNA repair. These responses are mediated by a kinase cascade that appears to be conserved among eukaryotes. Two essential genes, MEC1 and RAD53, are central players in the kinase cascade that leads to cell-cycle arrest at all the checkpoints and transcriptional activation in response to DNA damage. Cells with mutations in the Mec1 or Rad53 genes are defective in both cell-cycle arrest and gene expression responses in response to DNA damage, but retain their essential functions for cell survival under normal conditions [1][2][3][4][5]. Among the best characterized transcriptional targets of the Rad53 kinase cascade are the RNR genes, which encode subunits of ribonucleotide reductase, the enzyme that catalyzes the rate-limiting step in dNTP synthesis [1]. In addition to transcriptional induction of RNR genes, the Rad53 pathway also induces RNR activity through the removal of Sml1, an RNR inhibitor that binds to large subunits and inhibits enzyme activity [6]. Lethality of Rad53 null mutation is suppressed by overexpression of genes encoding ribonucleotide reductase [3] or by deletion of RNR inhibitor Sml1...

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Section: Discussionsupporting

confidence: 90%

“…It has been shown that cell survival after DNA damage is directly linked to elevated levels of dNTPs in yeast [11]. Mammalian cell response to DNA damage is somewhat ambiguous since neither logarithmically growing nor synchronized cells show accumulation of dNTP pools after DNA damage [12].…”

Section: Discussionmentioning

confidence: 99%

Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

Koç

Merrill

2007

Biochemical and Biophysical Research Communications

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“…The second is feedback inhibition by dATP. Chabes et al have recently shown that S. cerevisiae RNR is 10 times less sensitive to dATP inhibition than the mouse or calf thymus RNRs, requiring 50 μM dATP (with 5 mM ATP present) to observe inhibition in vitro (23,60). In our crude cell assays described above, the dNTPs have been removed by the ammonium sulfate fractionation and the Sml1 levels are very low due to its low concentrations inside the cell and relatively high K D with α (∼0.4 M) (74).…”

Section: Specific Activity Measurements Suggest That Rnr Need Not Be mentioning

confidence: 99%

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

et al. 2006

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The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y • ) cofactor. In Saccharomyces cerevisiae, two R1s, α n and , have been identified, while R2 is a heterodimer (ββ′). β′ cannot bind iron and generate the Y • ; consequently, the maximum amount of Y • per ββ′ is 1. To determine the cofactor stoichiometry in vivo, a FLAGtagged β ( FLAG β) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of FLAG ββ′ by immunoaffinity chromatography, which was found to have 0.45 ± 0.08 Y • / FLAG ββ′ and a specific activity of 2.3 ± 0.5 μmol min −1 mg −1 . FLAG ββ′ isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y • / FLAG ββ′ ratio of 0.5. To determine the Y • /ββ′ ratio without R2 isolation, whole cell EPR and quantitative Western blots of β were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y • /ββ′ ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Δ gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y • /ββ′ ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y • and modulation of Y • concentration is not involved in the regulation of RNR activity.

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“…The replication checkpoint response slows S-phase progression, stabilizes stalled replication forks, and increases RNR complex activity (Santocanale and Diffley, 1998;Lopes et al, 2001;Tercero and Diffley, 2001;Chabes et al, 2003). Recent data demonstrate that stabilization of replication forks is the main checkpoint function needed for cell survival in the face of replication stress.…”

Section: Introductionmentioning

confidence: 99%

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

Woolstencroft

Cook

Preiß

et al. 2006

Journal of Cell Science

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In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Δ dun1Δ strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Δ and chk1Δ exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Δ HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Δ dun1Δ strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

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Survival of DNA Damage in Yeast Directly Depends on Increased dNTP Levels Allowed by Relaxed Feedback Inhibition of Ribonucleotide Reductase

Cited by 390 publications

References 42 publications

Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

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