2′, 3′-Dideoxy-3′ amlnonudeo 5′ .triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases (original) (raw)
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Nucleic Acids Research
It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase a and rat liver DNA polymerase fi . The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.
Termination of DNA synthesis by novel 3'-modifieddeoxyribonucleoside 5'-triphosphates
Nucleic Acids Research, 1994
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-0-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-0-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stopstart DNA synthesis are initial steps in the development of a novel sequencing strategy.
FEBS Letters, 1993
S-Triphosphates of 1-(2',3'-epithio-2'.3'-dideoxy-B_D-lyxofuranosyl)thymine, l-(2',3'-eptthio-~,~-dideoxy-~-D-ribofuranosyl)th~lne and 2',3'lyxoanhydrothymidine have been shown to be termmatton substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase 1 from E. co11 and DNA polymerase j3 from rat hver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase E from human placenta. Km values of ItTTP. rtTTP and laTTP incorporation mto the DNA chain during catalysts by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than K,,, value for dTTP Furthermore, V,,, values for modified substrates are only 2-3 times lower than b',,,',,, for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides wtth a flattened furanosyl rmg for DNA polymerase active sites.
Journal of Biological Chemistry, 1995
5-Triphosphates of -D and -L-enantiomers of 2,3dideoxycytidine (ddC), 2,3-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases ␣, , ␥, ␦, and ⑀. L-ddCTP was not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA polymerases and was a less potent inhibitor of DNA polymerases ␥ and  than its D-enantiomer by 2 orders of magnitude. In contrast, all L-dioxolane analogs were potent inhibitors and chain terminators for all cellular DNA polymerases studied. The K i values of their 5-triphosphates for DNA polymerase ␥ were found to be in the following order: D-ddC < D-FddC L-OddC D-FOddC < L-FOddC < < L-FddC. The K i values of L-OddCTP for the reactions catalyzed by DNA polymerases ␣, ␦, ⑀, , and ␥ were 6.0, 1.9, 0.4, 3.0, and 0.014 M, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 M, respectively. The K m values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 M for DNA polymerases ␣, ␦, ⑀, , and ␥, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity.
FEBS Letters, 1987
dehydrothymidine 5'-triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase r, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3'-termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase ct. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.
3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis
FEBS Letters, 1985
It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase. The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase a. The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs. dNTP(3'F) produce a more clear sequence pattern than do ddNTPs.
Nucleoside 5′-triphosphates with modified sugars as substrates for DNA polymerases
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1986
A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase a. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates, ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase a only, but araNTP(3'NH2) was inactive with E. coil DNA polymerase I. dTTP(3'N3),
Nucleic Acids Research, 2003
A series of charge-modi®ed, dye-labeled 2¢, 3¢-dideoxynucleoside-5¢-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dyelabeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction puri®cation is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modi®ed nucleotides have also been shown to be more ef®ciently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5¢triphosphates.
Nucleic Acids Research, 2005
A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the a-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases a, b and l, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase b incorporated these derivatives in a strictly Mn 11 -dependent manner. On the other hand, polymerase l could incorporate some alkyltriphosphate derivatives with both Mg 11 and Mn 11 , but only opposite to an abasic site on the template strand. The active site mutant polymerase l Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.