Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion

Bourdon, Alice; Minai, Limor; Serre, Valérie; Jaı̈s, Jean-Philippe; Sarzi, Emmanuelle; Aubert, S.; Chrétien, Dominique; Lonlay, Pascale de; Paquis‐Flucklinger, Véronique; Arakawa, Hirofumi; Nakamura, Yusuke; Münnich, Arnold; Rötig, Agnès

doi:10.1038/ng2040

Cited by 468 publications

(407 citation statements)

References 24 publications

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“…The requirement reported here of mitochondrialto-nucleus retrograde signaling for dNTP pool homoeostasis in yeast may be of importance to a recent report that mutations in p53R2 cause human mitochondrial depletion syndromes (MDS) (40,41). If compensatory/ buffering relationships between RNR and retrograde signaling in yeast are evolutionarily conserved, then genetic variation in retrograde signaling may modulate MDS disease phenotypes resulting from deficiency in p53R2 activity.…”

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confidence: 63%

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Hartman

2007

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

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Synergistically interacting gene mutations reveal buffering relationships that provide growth homeostasis through their compensation of one another. This analysis in Saccharomyces cerevisiae revealed genetic modules involved in tricarboxylic acid cycle regulation (RTG1, RTG2, RTG3), threonine biosynthesis (HOM3, HOM2, HOM6, THR1, THR4), amino acid permease trafficking (LST4, LST7), and threonine catabolism (GLY1). These modules contribute to a molecular circuit that regulates threonine metabolism and buffers deficiency in deoxyribonucleotide biosynthesis. Phenotypic, genetic, and biochemical evidence for this buffering circuit was obtained through analysis of deletion mutants, titratable alleles of ribonucleotide reductase genes, and measurements of intracellular deoxyribonucleotide pool concentrations. This circuit provides experimental evidence, in eukaryotes, for the presence of a high-flux backbone of metabolism, which was previously predicted from in silico modeling of global metabolism in bacteria. This part of the high-flux backbone appears to buffer deficiency in ribonucleotide reductase by enabling a compensatory increase in de novo purine biosynthesis that provides additional rate-limiting substrates for dNTP production and DNA synthesis. Hypotheses regarding unexpected connections between these metabolic pathways were facilitated by genome-wide but also highly quantitative phenotypic assessment of interactions. Validation of these hypotheses substantiates the added benefit of quantitative phenotyping for identifying subtleties in gene interaction networks that modulate cellular phenotypes.genetic buffering ͉ high-flux backbone of metabolism ͉ protein trafficking ͉ ribonucleotide reductase ͉ mitochondria-to-nucleus retrograde signaling pathway C ells are complex genetic systems, having evolved compensatory molecular networks that provide growth homeostasis (robustness). Conceptually, gene interactions underlie robustness by buffering environmental or genetic perturbations (1-3). Synergistic effects on the phenotype resulting from two genetic deficiencies or chemical inhibition in combination with a genetic deficiency reveal buffering relationships when the double limitation is more severe than either single limitation. Genome-wide phenotypic analysis, as possible with RNAi or use of the complete set of yeast gene deletion mutants, has enabled new approaches to investigate buffering relationships systematically (2, 4, 5). It has been shown that quantitative (strength) and qualitative (pattern) aspects of gene interaction profiles reveal how genes organize in a pathway or cellular process (4, 6). Conceptually, such sets of genes represent genetic modules that contribute buffering capacity to the cell, providing insight into how molecular circuitry is arranged to achieve robustness (7,8). Comprehensive and quantitative methods for genotypephenotype analysis are becoming available for gaining a more global and precise understanding of buffering networks (4, 6, 9). These methods permit unbiased experimental...

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confidence: 63%

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Hartman

2007

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

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“…26), mitochondrial transcription factor Tfam 38 and ribonucleotide reductase subunit p53R2 (ref. 39). Maximum exercise capacity was shown to be impaired in p53 À / À mice.…”

Section: Discussionmentioning

confidence: 99%

Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart

et al. 2013

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Cumulative evidence indicates that mitochondrial dysfunction has a role in heart failure progression, but whether mitochondrial quality control mechanisms are involved in the development of cardiac dysfunction remains unclear. Here we show that cytosolic p53 impairs autophagic degradation of damaged mitochondria and facilitates mitochondrial dysfunction and heart failure in mice. Prevalence and induction of mitochondrial autophagy is attenuated by senescence or doxorubicin treatment in vitro and in vivo. We show that cytosolic p53 binds to Parkin and disturbs its translocation to damaged mitochondria and their subsequent clearance by mitophagy. p53-deficient mice show less decline of mitochondrial integrity and cardiac functional reserve with increasing age or after treatment with doxorubicin. Furthermore, overexpression of Parkin ameliorates the functional decline in aged hearts, and is accompanied by decreased senescence-associated b-galactosidase activity and proinflammatory phenotypes. Thus, p53-mediated inhibition of mitophagy modulates cardiac dysfunction, raising the possibility that therapeutic activation of mitophagy by inhibiting cytosolic p53 may ameliorate heart failure and symptoms of cardiac ageing.

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“…Thus our data do not support the suggestion that nuclear translocation is a new additional mechanism regulating ribonucleotide reduction in mammalian cells. The demonstration that the major function of p53R2 is to supply dNTPs for replication of mitochondrial DNA in postmitotic cells (20) has now shifted the focus from DNA repair to mitochondrial DNA synthesis. The new function of p53R2 makes its nuclear localization less likely and our present results indeed point in the same direction.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas an involvement of p53R2 in DNA repair has been difficult to demonstrate unequivocally, the discovery that children with severe mitochondrial DNA depletions carry functionally important mutations in p53R2 (20) clearly demonstrates the importance of p53R2 for mitochondrial DNA replication. Therefore it is now established that in vivo p53R2, in conjunction with R1, is required for mitochondrial DNA synthesis in differentiated tissues.…”

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confidence: 99%

Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

Pontarin¹,

Fijolek

Pizzo³

et al. 2008

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

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Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.DNA precursors ͉ immunofluorescence ͉ mitochondrial DNA ͉ p53R2 ͉ subcellular localization D NA replication and repair require a balanced supply of the four common deoxynucleoside triphosphates (dNTPs). In mammalian cells DNA synthesis occurs in two separate compartments: nucleus and mitochondria. The complete nuclear DNA is replicated only in cycling cells during S-phase, whereas cycling and quiescent cells replicate mitochondrial DNA and repair damaged DNA during their whole existence. Thus cycling cells require during a limited period a large supply of dNTPs in the nucleus. Outside S-phase cells consume much smaller amounts of dNTPs, mainly in the cytosol for mitochondrial (mt) DNA replication. In all cells the major supply of dNTPs comes from the de novo reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1).In cycling cells, the dominant form of mammalian RNR consists of two proteins called R1 and R2. The activity of the R1/R2 enzyme is exquisitely regulated by allosteric mechanisms involving nucleoside triphosphates and also by S-phase-specific transcription and proteasome-mediated degradation of R2 in late mitosis (2). Thus postmitotic cells are completely devoid of protein R2. How do these cells synthesize dNTPs for mitochondrial DNA replication and DNA repair? Until recently the answer to this question was by salvage of deoxynucleosides but the picture changed su...

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Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion

Cited by 468 publications

References 24 publications

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Buffering of deoxyribonucleotide pool homeostasis by threonine metabolism

Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart

Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

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