Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Intestinal alkaline phosphatase promotes gut bacterial growth by reducing the concentration of luminal nucleotide triphosphates.

The intestinal microbiota plays a pivotal role in maintaining human health and well-being. Previously, we have shown that mice deficient in the brush-border enzyme intestinal alkaline phosphatase (IAP) suffer from dysbiosis and that oral IAP supplementation normalizes the gut flora. Here we aimed to decipher the molecular mechanism by which IAP promotes bacterial growth. We used an isolated mouse intestinal loop model to directly examine the effect of exogenous IAP on the growth of specific intestinal bacterial species. We studied the effects of various IAP targets on the growth of stool aerobic and anaerobic bacteria as well as on a few specific gut organisms. We determined the effects of ATP and other nucleotides on bacterial growth. Furthermore, we examined the effects of IAP on reversing the inhibitory effects of nucleotides on bacterial growth. We have confirmed that local IAP bioactivity creates a luminal environment that promotes the growth of a wide range of commensal organisms. IAP promotes the growth of stool aerobic and anaerobic bacteria and appears to exert its growth promoting effects by inactivating (dephosphorylating) luminal ATP and other luminal nucleotide triphosphates. We observed that compared with wild-type mice, IAP-knockout mice have more ATP in their luminal contents, and exogenous IAP can reverse the ATP-mediated inhibition of bacterial growth in the isolated intestinal loop. In conclusion, IAP appears to promote the growth of intestinal commensal bacteria by inhibiting the concentration of luminal nucleotide triphosphates.

Effects of dNTPs on the SCE frequency in plant root tip cells.

The effects of deoxynucleoside triphosphate (dNTPs) on the frequency of sister-chromatid exchange (SCE) have been studied in plant root tip cells. Treatment with dATP caused a dose-dependent decrease in SCE frequency, while dGTP, caused a dose-dependent increase in SCE frequency both in Hordeum vulgare and in Vicia faba root tip cells. Treatment with dCTP and dTTP significantly increased SCE frequency in H. vulgare root tip cells, but significantly decreased SCE frequency in V. faba root tip cells. The SCE induced by treatment with dGTP could be totally reversed by treatment with an equal concentration of any of the other three dNTPs (dATP, dCTP and dTTP) both in H. vulgare and in V. faba. Treatment with a mixed equal concentration of dATP, dCTP, dGTP and dTTP did not alter the SCE frequency. These results suggest that dNTP pool imbalance is an SCE-affecting factor in plant cells.

Dietary nucleotides: effects on cell proliferation following partial hepatectomy in rats fed NIH-31, AIN-76A, or folate/methyl-deficient diets.

The requirement of a number of tissues for dietary nucleotides could explain some of the differences observed in animals fed natural ingredient diets vs. those fed purified diets lacking a source of dietary nucleotides. Lack of dietary nucleotides is exacerbated in animals fed folate- or methyl-deficient semipurified diets, in which both salvage and folate-dependent de novo synthetic pathways are diminished. We examined hepatocyte proliferation following partial hepatectomy in weanling male Fischer-344 rats fed natural ingredient NIH-31 diet, nucleotide-free purified AIN-76A diet or a basal diet similar to AIN-76A but deficient in the methyl donors folate, choline and methionine. Additional groups were fed AIN-76A or folate/methyl-deficient diets supplemented with 0.25% yeast RNA. Compared with NIH-31, AIN-76A increased dUMP/dTTP ratios, reduced the mitotic index (MI) and increased the ratio of proliferating cell index (PCI) to mitotic cells, an indication that hepatocytes were delayed in S-phase. Addition of yeast RNA to AIN-76A reversed (by approximately 50%) the effects of AIN-76A on dUMP/dTTP and cell proliferation. A folate/methyl-deficient diet also produced an increased dUMP/dTTP ratio and markedly reduced the MI, increasing the PCI/MI, which suggested even further delay of cells in S-phase. Addition of yeast RNA to the folate/methyl-deficient diet was effective in significantly reversing the effects of folate/methyl deficiency.

Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.

Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.

Synthesis and antiviral activity evaluation of 3'-fluoro-3'-deoxyribonucleosides: broad-spectrum antiviral activity of 3'-fluoro-3'-deoxyadenosine.

Five 3'-fluorinated ribonucleosides were prepared and evaluated for their inhibitory properties against different viruses. The synthesis of these compounds was achieved by treatment of 2',5'-di-O-tritylated nucleoside analogues possessing a xylo-configuration with diethylaminosulfur trifluoride, followed by deprotection. 3'-Fluoro-3'-deoxyadenosine was active against a broad range of viruses, encompassing both DNA viruses [pox (vaccinia)], single-stranded (+) RNA viruses [picorna (polio, Coxsackie B), toga (sindbis, Semliki Forest)] and double-stranded RNA viruses (reo). In its antiviral activity spectrum 3'-fluoro-3'-deoxyadenosine clearly differed from those adenosine analogues that are known as inhibitors of S-adenosylhomocysteine hydrolase. 3'-Fluoro-3'-deoxyadenosine also proved effective in vivo, in inhibiting tail lesion formation in mice inoculated intravenously with vaccinia virus.

Complexes of Rep protein with ATP and DNA as a basis for helicase action.

The rep protein of Escherichia coli, a helicase that unwinds duplex DNA at a replication fork (Kornberg, A., Scott, J. F., and Bertsch, L. L. (1978) J. Biol. Chem. 253, 3298-3304), forms binary complexes with ATP and with DNA, and ternary complexes with both. ATP (or dATP) is bound at a single site with a dissociation constant (KD) near 10(-7) M. Other ribonucleoside triphosphates and deoxyribonucleoside triphosphates compete for the same site with far lower affinities. The protein forms a binary complex with single-stranded DNA and with duplex DNA, each at distinctive sites. Binding to single-stranded DNA covers a stretch of approximately 20 nucleotides, destabilizes secondary structure, and facilitates reannealing of complementary single strands. Ternary complexes of rep protein with ATP and DNA are manifested by ATP hydrolysis and by binding of labeled components. Nonhydrolyzed ATP analogs are useful aids for isolation and studies of such complexes. Unlike rep protein's processive action as a helicase at a replication fork, its action on single-stranded DNA is distributive, with ATP hydrolysis accelerating dissociation of the protein from the complex. These and related studies serve as guides to understanding the multiple interactions of rep protein with its ATP and DNA ligands that enable it to unwind duplex DNA at a replication fork.

In vitro synthesis of infectious transforming DNA by the avian sarcoma virus reverse transcriptase.

Infectious DNA molecules, capable of transforming chicken embryo fibroblasts, can be synthesized by the Rous sarcoma virus-associated reverse transcriptase in vitro. The optimal enzymatic conditions employed for infectious DNA synthesis also facilitate maximum synthesis of genome length DNA. Analysis of the DNA product synthesized by detergent-disrupted Rous sarcoma virus under these conditions indicates that DNA complementary to viral RNA (minus-strand DNA) is genome length in size, whereas DNA complementary to genome length minus-strand DNA (plus-strand DNA) appears as subgenomic-length molecules ranging between 300 and 3,500 nucleotides in length. These features of the DNA product synthesized by the Rous sarcoma virus reverse transcriptase in vitro are similar to those identified in the cytoplasm of cells shortly after infection and lend credence to studies of the mechanism of reverse transcription in vitro and their significance to proviral DNA synthesis in vivo.