The DNA Replication and Damage Checkpoint Pathways Induce Transcription by Inhibition of the Crt1 Repressor

Huang, Mingxia; Zhou, Zheng; Elledge, Stephen J.

doi:10.1016/s0092-8674(00)81601-3

Cited by 474 publications

(689 citation statements)

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“…11 This mechanism of R2 repression was first identified in yeast where the Rfx1 ortholog, Crt1, represses the R2 gene. 29 Consistent with this repression role of Rfx1, chromatin immunoprecipitation (ChIP) analysis revealed that Rfx1 was bound to the R2 promoter more effectively in the quiescent cells than in the proliferative cells (Fig. 6A).…”

Section: Resultssupporting

confidence: 58%

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10 7 to 10 11 virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene. Conclusion: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs. (HEPATOLOGY 2010;51:1538-1546 H epatitis B virus (HBV) is a widespread pathogen responsible for acute and chronic hepatitis and is a causative factor of hepatocellular carcinoma (HCC). 1 HBV is a small-enveloped noncytopathic virus containing a circular partially doublestranded DNA genome, and exhibits strong tropism for human liver cells. 2 During replication, the viral polymerase reverse-transcribes the pregenomic RNA to DNA using deoxyribonucleotide triphosphates (dNTPs). HBV preferentially replicates in nondividing cells, 3 in which the concentration of dNTP is low, which raised the question whether dNTP concentration is adequate to support viral yield. The level of dNTPs in a nondividing adult liver cell is <0.4 lM. 4 The Michaelis constant (K m ) of the viral polymerase at a dNTP concentration of 0.4 lM is only 62%, whereas a 4 lM concentration gives 93.3% activity. 5 Furthermore, one hepatocyte produces 50-300 hepatitis B virions per day, 6 and because the HBV genome is approximately 3.2 kilobases, between 3 Â 10 5 to 2 Â 10 6 dNTPs are consumed per day in this process. Considering cell volume as 500 fL, 7 a resting cell contains approximately 1.2 Â 10 5 dNTP molecules. Thus, the total amount of dNTPs used for HBV production per day exceeds the amount found in a nondividing hepatocyte. Because HBV does not activate the cell cycl...

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Section: Resultssupporting

confidence: 58%

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Cohen¹,

Adamovich²,

Reuven³

et al. 2009

Hepatology

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“…This organism possesses no deoxynucleoside kinase activities, and thus, dNTP synthesis is entirely dependent on RNR (72). In S. cerevisiae, the levels of the subunits are controlled transcriptionally and the level of mRNA of the α subunit is increased during the S-phase of the cell cycle during normal growth, while the levels of mRNA for the β and β′ subunits remain largely unchanged (22,25,27,31). We have recently shown that the mRNA levels are correlated with the protein levels (D. L. Perlstein, M. Huang, and J. Stubbe, unpublished results).…”

Section: Discussionmentioning

confidence: 94%

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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“…Sml1 is phosphorylated by Dun1 and subsequently targeted for proteolysis, which relieves physical inhibition of RNR activity (Zhao and Rothstein, 2002). Crt1 inhibits transcription at target promoters through recruitment of the general transcriptional repressors Tup1 and Ssn6 (Huang et al, 1998;Li and Reese, 2001). Notably, Crt1 is phosphorylated in a Mec1-Rad53-Dun1-dependent manner after DNA damage or replication stress, thereby promoting its dissociation from promoter DNA and transcriptional activation (Huang et al, 1998).…”

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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The DNA Replication and Damage Checkpoint Pathways Induce Transcription by Inhibition of the Crt1 Repressor

Cited by 474 publications

References 29 publications

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene

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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