title: Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates.
abstract: The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-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 is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.
authors.affiliation: Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Shuhei
authors.initials: S
authors.lastname: Imoto
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Patro
authors.forename: Yu Lin
authors.initials: YL
authors.lastname: Jiang
authors.forename: Natsuhisa
authors.initials: N
authors.lastname: Oka
authors.forename: Marc M
authors.initials: MM
authors.lastname: Greenberg
mesh_terms: D001483:Base Sequence; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D006868:Hydrolysis; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*
chemical_list: D009711:Nucleotides; D010710:Phosphates; D011743:Pyrimidines; D004259:DNA-Directed DNA Polymerase