Silencing of TK2 and dGK affected the size of the four dNTP pools in myotubes (C). Surprisingly, all four dNTP pools became significantly smaller. The specificity of the involved kinases suggests that silencing of TK2 would affect the dTTP and possibly the dCTP pool and silencing of dGK the size of the dGTP and possibly the dATP pool (, ). Instead, the silencing of either enzyme activity decreased all four pool sizes with roughly similar efficiency (C). The effect of p53R2 silencing was smaller (D), in agreement with the marginal effect on mtDNA but consisted in a significant reduction of the dCTP and dGTP pools, as was observed in p53R2-mutated quiescent human fibroblasts (). The generalized decrease of dNTP pool sizes in differentiating myotubes silenced for TK2 or dGK was puzzling. We hypothesized that the depletion of the unexpected dNTPs, e.g. dGTP and dATP in the TK2-silenced myotubes, may be a secondary effect of the depletion of the expected dNTPs, in the case of TK2 silencing primarily dTTP. To test our hypothesis, we incubated the TK2-silenced cultures with CdR during 4 days of differentiation in DM. The labeling experiments in had shown that CdR is incorporated into both the dCTP and the dTTP pool, without altering their pool sizes. To obtain an increase of the dTTP and dCTP pool, we had to use 500 μ CdR, because concentrations between 5 and 100 μ were ineffective. The activity of catabolic enzymes kept the dCTP pool under control. With 500 μ CdR, we observed an expansion of the pyrimidine pools in both control and TK2-silenced myotubes (A), lower in the latter that could only phosphorylate CdR by deoxycytidine kinase, due to TK2 silencing. The increase in pyrimidine dNTPs was accompanied in the TK2-silenced myotubes by a normalization of the dATP pool and a partial recovery of the dGTP pool (B) and the mtDNA depletion (C).
Phosphoramidites that allow the generation of oligonucleotides containing site-specific lesions have been vital components for studying the mechanism of DNA repair. New DNA lesions are still being discovered and the study of their biological consequences will require their site-specific incorporation into oligonucleotides. The authors conclude that the increased availability of phosphoramidites for the synthesis of lesion-containing oligonucleotides should facilitate many future discoveries in the broad area of DNA damage and repair.
OligoAnalyzer 3.1 (Integrated DNA Technologies)
We report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the dNaM-dTPT3 UBP and its mispairs in a variety of sequence contexts. The data demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.
What Causes Aging? Damage-Based Theories of Aging
The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3'side, reading the template from 3' to 5' side, bases are added complementary to the template) [9-11].
Reverse transcription polymerase chain reaction - Wikipedia
A number of parameters of mt biogenesis have been monitored during muscle differentiation (, , ). However, the changes taking place in dNTP biosynthesis and pool sizes during early steps of muscle differentiation have not been examined in detail. Few of the genes coding for the relevant enzymes vary above the threshold of microarray expression analyses and the low amounts of the individual proteins escape detection in proteomic surveys (–). Enzyme assays in protein extracts from animal tissues () may be biased by the presence in the tissues of multiple cell types. Thus, the features underlying the distinctive sensitivity of skeletal muscle to genetic defects of enzymes of dNTP metabolism are only incompletely understood, and the consequences of the enzyme deficiencies for the availability of precursors during mt biogenesis are largely unknown. Determination of the dNTP pool sizes of muscle fibers in vivo is technically challenging and impracticable in the case of patients with severe myopathies ().
Arbuscular mycorrhiza - Wikipedia
We have chosen cultures of C2C12 murine myoblasts to study how the enzyme network regulating dNTP synthesis changes during the initial phases of myogenesis and how a down-regulation of enzymes involved in mtDNA depletion syndromes affects the differentiation of myotubes and the expansion of mtDNA. Differentiated cultures of C2C12 cells contain a significant residual fraction of mononucleated cells whose dNTP pool sizes and enzymatic setup differ from those of differentiated myotubes. To avoid the confounding influence of non-fused cells, we devised a protocol for the purification of differentiated myotubes. In proliferating myoblasts, differentiated cultures, and isolated myotubes, we compared the expression and activities of key enzymes of dNTP synthesis and the size of the four dNTP pools, demonstrating a low abundance of dNTPs in myotubes and a low expression of their biosynthetic pathways. By siRNA transfection, we individually down-regulated TK2, dGK, or p53R2 to examine how a further reduction of synthetic activities impacts the development of multinucleated myotubes.