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Ribonucleotide Reductase Association with Mammalian Liver Mitochondria
Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly
asymmetric, and this asymmetry probably contributes toward the elevated
mutation rate for the mitochondrial genome as compared with the nuclear
genome. To understand this asymmetry, we must identify pathways for synthesis
and accumulation of dNTPs within mitochondria. We have identified
ribonucleotide reductase activity specifically associated with mammalian tissue
mitochondria. Examination of immunoprecipitated proteins by mass spectrometry
revealed R1, the large RNR subunit, in purified mitochondria. Significant
enzymatic and immunological activity was seen in rat liver mitochondrial
nucleoids, isolated as described by Wang, Y., and Bogenhagen, D. F. (2006) J.
Biol. Chem. 281, 25791â25802. Moreover, incubation of respiring rat liver
mitochondria with [ÂčâŽC]cytidine diphosphate leads to acccumulation of
radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria.
Comparable results were seen with [ÂčâŽC]guanosine diphosphate. Ribonucleotide
reduction within the mitochondrion, as well as outside the organelle, needs to be
considered as a possibly significant contributor to mitochondrial dNTP pools.This research was originally published in the Journal of Biological Chemistry. Chimploy, K., Song, S., Wheeler, L. J., & Mathews, C. K. Ribonucleotide reductase association with mammalian liver mitochondria. 2013. 288(18), 13145-13155. © the American Society for Biochemistry and Molecular Biology. This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the American Society for Biochemistry and Molecular Biology and can be found at: http://www.jbc.org/.Keywords: deoxyribonucleotide metabolism, ribonucleotide reductase, mitochondria, nucleotide pool asymmetryKeywords: deoxyribonucleotide metabolism, ribonucleotide reductase, mitochondria, nucleotide pool asymmetr
DNA building blocks: keeping control of manufacture
Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α6ÎČ2 complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved
Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase
Association of genetic polymorphisms in genes involved in Ara-C and dNTP metabolism pathway with chemosensitivity and prognosis of adult acute myeloid leukemia (AML)
Abstract Background Cytarabine arabinoside (Ara-C) has been the core of chemotherapy for adult acute myeloid leukemia (AML). Ara-C undergoes a three-step phosphorylation into the active metabolite Ara-C triphosphosphate (ara-CTP). Several enzymes are involved directly or indirectly in either the formation or detoxification of ara-CTP. Methods A total of 12 eQTL (expression Quantitative Trait Loci) single nucleotide polymorphisms (SNPs) or tag SNPs in 7 genes including CMPK1, NME1, NME2, RRM1, RRM2, SAMHD1 and E2F1 were genotyped in 361 Chinese non-M3 AML patients by using the Sequenom Massarray system. Association of the SNPs with complete remission (CR) rate after Ara-C based induction therapy, relapse-free survival (RFS) and overall survival (OS) were analyzed. Results Three SNPs were observed to be associated increased risk of chemoresistance indicated by CR rate (NME2 rs3744660, E2F1 rs3213150, and RRM2 rs1130609), among which two (rs3744660 and rs1130609) were eQTL. Combined genotypes based on E2F1 rs3213150 and RRM2 rs1130609 polymorphisms further increased the risk of non-CR. The SAMHD1 eQTL polymorphism rs6102991 showed decreased risk of non-CR marginally (Pâ=â0.055). Three SNPs (NME1 rs3760468 and rs2302254, and NME2 rs3744660) were associated with worse RFS, and the RRM2 rs1130609 polymorphism was marginally associated with worse RFS (Pâ=â0.085) and OS (Pâ=â0.080). Three SNPs (NME1 rs3760468, NME2 rs3744660, and RRM1 rs183484) were associated with worse OS in AML patients. Conclusion Data from our study demonstrated that SNPs in Ara-C and dNTP metabolic pathway predict chemosensitivity and prognosis of AML patients in China