Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates
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- 作者：Shuhei Imoto ; Jennifer N. Patro ; Yu Lin Jiang ; Natsuhisa Oka ; Marc M. Greenberg
- 刊名：Journal of the American Chemical Society
- 出版年：2006
- 出版时间：November 15, 2006
- 年：2006
- 卷：128
- 期：45
- 页码：14606 - 14611
- 全文大小：143K
- 年卷期：v.128,no.45(November 15, 2006)
- ISSN：1520-5126
文摘
The nucleoside triphosphates of N6-(2-deoxy-
,
-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carriedout using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. TheKlenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and -C-Fapy·dGTP assubstrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modifiednucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. Thespecificity constants are sufficiently large that nucleoside triphosphate incorporation could account for thelevel of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of -C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes isenhanced by inefficient hydrolysis of Fapy·dGTP and -C-Fapy·dGTP by MutT, the E. coli enzyme thatreleases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurallyrelated nucleoside triphosphates.
,-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carriedout using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. TheKlenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and -C-Fapy·dGTP assubstrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modifiednucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. Thespecificity constants are sufficiently large that nucleoside triphosphate incorporation could account for thelevel of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of -C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes isenhanced by inefficient hydrolysis of Fapy·dGTP and -C-Fapy·dGTP by MutT, the E. coli enzyme thatreleases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurallyrelated nucleoside triphosphates.