Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of <i>mec1</i> and<i>rad53</i> Lethality

Zhao, Xiaolan; Georgieva, Bilyana; Chabes, Andrei; Domkin, V. D.; Ippel, Hans; Schleucher, Jürgen; Wijmenga, Sybren S.; Thelander, Lars; Rothstein, Rodney

doi:10.1128/mcb.20.23.9076-9083.2000

Cited by 84 publications

(113 citation statements)

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“…1A and B). Residues 28 to 50 of Sml1 were predicted to be a nonstructural linker region between the N-and C-terminal alpha-helical regions of the protein; removal of this region does not affect Sml1's inhibition of RNR activity (53). We found that removal of residues 28 to 50 in Sml1 greatly increased stability of the protein both in cells under normal growth conditions and in cells treated with HU and MMS (Fig.…”

Section: Resultsmentioning

confidence: 55%

“…Sml1 is an unstable protein and undergoes checkpoint-dependent phosphorylation and degradation during S phase of the cell cycle and in cells experiencing genotoxic stress (52,55). Although no apparent sequence homolog of Sml1 has been identified in multicellular organisms, Sml1 can bind the mammalian R1 and inhibits its activity (8,53), suggesting a conserved mechanism of Sml1-R1 interaction and inhibition.…”

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confidence: 99%

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Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

Huang

2008

Molecular and Cellular Biology

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Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase (RNR). RNR is controlled via transcription, protein inhibitor association, and subcellular localization of its two subunits, R1 and R2. Saccharomyces cerevisiae Sml1 binds R1 and inhibits its activity, while Schizosaccharomyces pombe Spd1 impedes RNR holoenzyme formation by sequestering R2 in the nucleus away from the cytoplasmic R1. Here we report the identification and characterization of S. cerevisiae Dif1, a regulator of R2 nuclear localization and member of a new family of proteins sharing separate homologous domains with Spd1 and Sml1. Dif1 is localized in the cytoplasm and acts in a pathway different from the nuclear R2-anchoring protein Wtm1. Like Sml1 and Spd1, Dif1 is phosphorylated and degraded in cells encountering DNA damage, thereby relieving inhibition of RNR. A shared domain between Sml1 and Dif1 controls checkpoint kinase-mediated phosphorylation and degradation of the two proteins. Abolishing Dif1 phosphorylation stabilizes the protein and delays damage-induced nucleus-to-cytoplasm redistribution of R2. This study suggests that Dif1 is required for nuclear import of the R2 subunit and plays an essential role in regulating the dynamic RNR subcellular localization.Maintenance of genomic stability depends on faithful replication of DNA and repair of lesions after damage. Fidelity of both DNA replication and repair is influenced by perturbation in the sizes and relative ratios of cellular deoxynucleotide triphosphate (dNTP) pools. Ribonucleotide reductase (RNR) catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms and is largely responsible for maintaining cellular dNTP pools (34). As maximal DNA synthesis requires high concentrations of dNTPs, the RNR activity plays an important role in cell proliferation (31). On the other hand, increased RNR activity has been associated with malignant transformation and resistance to chemotherapy (12,14,25,56).The class I RNR holoenzymes are commonly found in eukaryotes and eubacteria and comprise two subunits, R1 and R2 (34). The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42). Mammalian genomes contain a single R1 gene and two R2 genes; the cell cycle-regulated RRM2 is responsible for providing dNTPs in actively dividing cells, and the DNA damage-inducible p53R2 is required for replenishing dNTP pools in cells under genotoxic stress (9,22,44). Loss of p53R2 causes mitochondrial DNA depletion and increased apoptosis (22). The budding yeast Saccharomyces cerevisiae has two R1 genes, RNR1 and RNR3. RNR1 is essential for mitotic growth, while RNR3 is high...

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Section: Resultsmentioning

confidence: 55%

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confidence: 99%

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

Huang

2008

Molecular and Cellular Biology

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“…At present, there are three mechanisms proposed to regulate RNR activity. One involves the small protein Sml1 that binds to α and causes inhibition by a poorly understood mechanism (74,78,79). The second is feedback inhibition by dATP.…”

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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“…Measurement of the frequency of petite formation and the two-hybrid assay method has been described (3). Cell cycle arrest, fluorescence-activated cell sorting, genotoxin treatment, extraction of yeast proteins, and protein blot analysis have been described (5,19). The IMAGEQUANT program (Molecular Dynamics) was applied to measure the density of bands on protein blots.…”

Section: Dun1mentioning

confidence: 99%

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

Zhao

Rothstein²

2002

Proc. Natl. Acad. Sci. U.S.A.

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Cell cycle checkpoints are evolutionarily conserved surveillance systems that protect genomic stability and prevent oncogenesis in mammals. One important target of checkpoint control is ribonucleotide reductase (RNR), which catalyzes the rate-limiting step in dNTP and DNA synthesis. In both yeast and humans, RNR is transcriptionally induced after DNA damage via Mec1͞Rad53 (yeast) and ATM͞CHK2 (human) checkpoint pathways. In addition, yeast checkpoint proteins Mec1 and Rad53 also regulate the RNR inhibitor Sml1. After DNA damage or at S phase, Mec1 and Rad53 control the phosphorylation and concomitant degradation of Sml1 protein. This new layer of control contributes to the increased dNTP production likely necessary for DNA repair and replication; however, the molecular mechanism is unclear. Here we show that Dun1, a downstream kinase of Mec1͞Rad53, genetically and physically interacts with Sml1 in vivo. The absence of Dun1 activity leads to the accumulation of Sml1 protein at S phase and after DNA damage. As a result, dun1⌬ strains need more time to finish DNA replication, are defective in mitochondrial DNA propagation, and are sensitive to DNA-damaging agents. Moreover, phospho-Sml1 is absent or dramatically reduced in dun1⌬ cells. Finally, Dun1 can phosphorylate Sml1 in vitro. These results suggest that Dun1 kinase function is the last step required in the Mec1͞Rad53 cascade to remove Sml1 during S phase and after DNA damage. R ibonucleotide reductase (RNR) catalyzes the conversion of NDP to dNDP, the rate-limiting step of dNTP synthesis. RNR activity directly affects the levels and balances of the dNTP pools and subsequently both genetic fidelity and cell viability (1).As an important enzyme in the early stages of DNA synthesis, RNR activity is tightly regulated during DNA replication and repair. Besides its intrinsic allosteric regulation, RNR activity is regulated at the transcriptional level (2) and also by its inhibitor, Sml1 (3, 4). In yeast, these last two types of control both require the evolutionarily conserved checkpoint kinases, Mec1 and Rad53 (5).In response to DNA damage, Mec1 and Rad53 activate the downstream checkpoint kinase Dun1, which leads to the transcriptional induction of the RNR genes (6-8). Such induction is achieved by the relief of transcriptional repression by the Crt1 protein (9). However, the direct downstream substrate(s) of the Dun1 kinase remain unknown. Similar transcriptional induction of the RNR genes also exists in mammals. For example, a human RNR small subunit (p53R2) is induced by p53 after DNA damage, and this induction is crucial for DNA repair (10). Given that the p53 is activated after DNA damage by the Mec1 and Rad53 homologs, ATM͞ATR and CHK2 (11), it is likely that the RNR transcriptional induction circuitry is largely conserved between yeast and mammals.In addition to transcriptional induction of the RNR genes, the Mec1͞Rad53 pathway also increases RNR activity by regulating the removal of the RNR inhibitor, Sml1 (3, 4). Sml1 is a 104-residue peptide that binds t...

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Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 andrad53 Lethality

Cited by 84 publications

References 36 publications

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

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

The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1

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