Deoxynucleotide triphosphates (dNTPs) are the nucleoside triphosphates containing deoxyribose. They are the building blocks of DNA, and they lose two of the phosphate groups when incorporated into DNA during replication. Members of dNTPs include deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxyuridine triphosphate (dUTP).
Besides abundant dNTPs such as dATP, dTTP, dCTP, dGTP and dUTP, some less abundant NTPs, such as the tautomeric forms of some dNTPs or artificial nucleotides are available. The tautomeric forms of dNTPs may lead to the mismatch during DNA replication. One example is that the tautomeric form of cytosine is capable of forming 3 hydrogen bonds with adenine and lead to a mismatch. Another example is that the deamination of 5-methylcytosine commonly encountered in eukaryotes will result in thymine. Those mismatches are not without being repaired. The 3' to 5' exonuclease activity of DNA polymerase III will excise the mismatched bases during replication and catalyzes the correct dNTPs being added.
4 kinds of deoxyribonucleoside triphosphates (dNTPs), dATP, dTTP, dCTP, dGTP are required for DNA replication and repair. The delicate balance of these 4 deoxyribonucleoside triphosphates is important for the correct synthesis of DNA. The four deoxyribonucleoside triphosphates (dNTPs) existing in eukaryotic cells are in minute amounts and sufficient for only a few minutes of DNA replication. Either a deficiency or a surplus of a single dNTP may result in increased mutation rates in DNA replication. The substrates for DNA polymerizing enzymes, dNTPs is limited in quantity in cells because ribonucleotide reductase (RNR), an enzyme that catalyze the conversion from ribonucleotides to deoxynucleotides, is synthesized and enzymatically activated only when cells enter the S phase. RNR, which is discovered 52 years ago, catalyze the formation deoxynucleoside disposphates (dNDPs) from four respective ribonucleotide diphosphates (rNDPs). The dNDPs in turn are rapidly converted to dNTPs. RNR levels and activity are rather low in noncycling cells and during the G1 phase of the cell-division cycle, while are dramatically increased when cells enter S phase of the cell division cycle or during DNA repair. Actually, RNR is well known as one of the most highly regulated enzymes. In mammals, this enzyme is allosterically activated by dATP, dTTP, and dGTP and is feed-back–inhibited by dATP. Other regulation factors include specific inhibitory proteins in yeasts, by cell cycle-dependent transcription of the genes and by RNR subunit protein stability.
Four dNTPs in eukaryotic cells are in rather low concentrations and undergoes large variations within different periods of the cell cycle. When cells stop cycling, the size of the dNTP pools shrink and is correspond to less than 10% of the sizes during cell replication, reaching as low as 1 pmol per million cells. Therefore, a sensitive and reliable quantification method for limited amounts of dNTPs in cell extracts becomes necessary to detect changes in the dNTP pools in genetically manipulated cells and animal models of human diseases and understand the mechanisms of nuclear and mitochondrial DNA replication and repair mutagenesis and apoptosis. Usually, dNTPs in cell extracts are quantified by two independent methods: high-performance liquid chromatography (HPLC) and enzymatic assay with DNA polymerase. Ideally, it’s better to combine these two methods because either method has its disadvantages. The highly sensitive LC-MS/MS methods were introduced for the quantification of dNTPs and cyclic-di-AMP recently.
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