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Fapy.dG and Fapy.dA are biologically interesting lesions that readily anomerize in aqueous solution, an unusual property for nucleotides. Experiments suggest that Fapy.dG exists almost exclusively as its Beta-anomer, and that Beta-Fapy.dA is also the major stereoisomer present in duplex DNA.

A study was conducted to analyze human DNA polymerase [alpha], a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF). Results indicate that different polymerases interact very differently with base lesions.

A series of dATP and dGTP analogues were used to determine the kinetics of DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase which used N-1, [N.sup.2], N-3, and [N.sup.6] of purine dNTPs to differentiate between right and wrong nucleotide incorporation. The results indicated that BF used N-1, [N.sup.2], N-3, and [N.sup.6] of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation.

The effect of Fapy.dG on replication in Escherichia coli is analyzed by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in local sequence contexts. The results have shown that a low dA misincorporation frequency opposite Fapy.dG and inefficient extension of a Fapy.dG:dA base pair have worked synergistically to minimize the levels of G -> T transversions.

The nucleoside triphosphates of N6-(2-deoxy-[alpha],[beta],D--erthro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTAP) and its C-nucleoside analogue ([beta]-C-Fapy.dGTP) are synthesized. 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.

An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine or 5'-deoxycytidine are prepared and employed in oligonucleotide synthesis.

We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N2 of a purine plays during dNTP polymerization by human DNA polymerase a. Removing N2 from dGTP had small effects during correct incorporation opposite C but specifically increased misincorporation opposite A. Adding N2 to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N2 greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N2 to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N2 and pyrimidine O2. Even in those cases where formation of a wobble base pair, tautomerization, and/or protonation of the base pair between the incoming dNTP and template base cannot occur (e.g., 2-pyridone purine (or purine analogue) base pairs), N2 enhanced formation of the base pair. Importantly, N2 had similar effects on dNTP polymerization both when added to the incoming purine dNTP and when added to the template base being replicated. The mechanistic implications of these results regarding how pol a discriminates between right and wrong dNTPs are discussed. [ABSTRACT FROM AUTHOR]

Fapy.dG (N(6)()-(2-deoxy-alpha,beta-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) is a modified purine lesion produced by a variety of DNA-damaging agents, which shows interesting biochemical properties. The previous method for synthesizing oligonucleotides containing Fapy.dG utilized a reverse dinucleotide phosphoramidite, which also required the synthesis of the appropriate reverse phosphoramidites. An improved method for synthesizing oligonucleotides containing Fapy.dG, which does not require reverse phosphoramidites, is described. Fapy.dG containing dinucleotide phosphoramidites containing 5'-thymidine (11a) or 5'-deoxycytidine (15) are prepared and employed in oligonucleotide synthesis. Oligonucleotide purity is assayed using the DNA repair enzyme formamidopyrimidine DNA glycosylase and by ESI-MS.