2′,3′-Dideoxynucleoside triphosphates (ddNTP) and di-2′,3′-dideoxynucleoside tetraphosphates (ddNp4ddN) behave differently to the corresponding NTP and Np4N counterparts as substrates of firefly luciferase, dinucleoside tetraphosphatase and phosphodiesterases1Dedicated to our friend, Professor Claudio Fernández de Heredia, on his 65th birthday.1

Sillero, Marı́a A. Günther; Madrid, Olga; Zaera, E.; Sillero, Antonio

doi:10.1016/s0304-4165(96)00092-x

Cited by 18 publications

(19 citation statements)

References 29 publications

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“…By contrast, dinucleoside polyphosphates (Np n NЈ) should be sensitive to cleavage by SVPD. Initial cleavage should produce Np and NЈp nϪ1 or Np nϪ1 and NЈp, and further digestion should convert all products to nucleoside monophosphates (NMP and NЈMP) (27,30). As expected, SVPD digestion of the labeled products formed from 32 Plabeled ddATP and GTP by firefly luciferase caused the Gp 4 ddA band to disappear and caused bands migrating as ddATP and ddAMP to increase (Fig.…”

Section: Characteristics Of a Nucleotide-dependent Primer Modifying Asupporting

confidence: 54%

“…The rate of synthesis of dinucleoside polyphosphates is limited by the intrinsic synthesis activity of the RT͞template-primer complex and the nucleotide concentration. The apparent k cat and K m were determined from the rate of specific product formation as a function of nucleotide concentration by using SIGMAPLOT (27). Enzyme Digestion of Primer Modification Products.…”

Section: Methodsmentioning

confidence: 99%

“…We used the reaction catalyzed by firef ly luciferase (27,29) with [␣-32 P]ddATP and unlabeled ATP or GTP to synthesize authentic labeled Ap 4 ddA (Fig. 2A, lane 2) or Gp 4 ddA (Fig.…”

Section: Characteristics Of a Nucleotide-dependent Primer Modifying Amentioning

confidence: 99%

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Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Meyer

Matsuura

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

278

331

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HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3 end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri-or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (K i Ϸ 5 M), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3 nucleotide from the primer to the pyrophosphate moiety in the nucleoside dior triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

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Section: Characteristics Of a Nucleotide-dependent Primer Modifying Asupporting

confidence: 54%

Section: Methodsmentioning

confidence: 99%

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Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Meyer

Matsuura

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

278

331

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“…Inorganic pyrophosphatase (EC 3.6.1.1) and alkaline phosphatase (grade I) (EC 3.1.3.1) were obtained from Boehringer Mannheim. Dinucleoside tetraphosphatase (EC 3.6.1.17) was purified from rat liver as previously described [38], Thin layer chromatographic (TLC) silica gel fluorescent plates were from Merck. For autoradiography X-ray films from Konica Corporation (Japan) were used.…”

Section: Methodsmentioning

confidence: 99%

“…The radioactivity under the ATP spot was gradually displaced towards new radioactive compounds corresponding, presumably, to p.IA and ApaG (Fig. 2), The nature of the newly synthesized radioactive products was assessed as p4A and Ap~G by the following criteria: comigration (TLC) or coelution (HPLC) with standards; treatment with alkaline phosphatase; treatment of the radioactive [adenylate-32p]AppppG with purified rat liver dinucleoside tetraphosphatase [30,38] which gave radioactive chromatographic spots corresponding to [o~5~2p]AMP and [c~-:~2p]ATP (Fig. 2c).…”

Section: Synthesis Q/p4a and Apjg Catalyzed B) T4 Dna Ligasementioning

confidence: 99%

T4 DNA ligase synthesizes dinucleoside polyphosphates

et al. 1998

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T4 DNA iigase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of ApIG and, conversely, tripolyphosphate (or GTP) inhibits the iigation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"-Pa,pa-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

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Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

Hacker

Welter

Marx

2015

CP Nucleic Acid Chemistry

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This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

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Cited by 18 publications

References 29 publications

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

T4 DNA ligase synthesizes dinucleoside polyphosphates

Synthesis of γ‐Phosphate‐Labeled and Doubly Labeled Adenosine Triphosphate Analogs

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