Rational attempts to optimize non-natural nucleotides for selective incorporation opposite an abasic site.

Abstract:

Translesion DNA synthesis represents the ability of a DNA polymerase to misinsert a nucleotide opposite a DNA lesion. Previous kinetic studies of the bacteriophage T4 DNA polymerase using a series of non-natural nucleotides suggest that pi-electron density of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site, a nontemplating DNA lesion. However, it is surprising that these nonhydrogen-bonding analogues can also be incorporated opposite natural templating DNA with variable degrees of efficiency. In this article, we describe attempts to achieve selectivity for incorporation opposite the abasic site through optimization of pi-electron density and shape of the nucleobase. Toward this goal, we report the synthesis and enzymatic characterization of two novel nucleotide analogues, 5-napthyl-indolyl-2'-deoxyriboside triphosphate (5-NapITP) and 5-anthracene-indolyl-2'-deoxyriboside triphosphate (5-AnITP). The overall catalytic efficiency for their incorporation opposite an abasic site is similar to that of other non-natural nucleotides such as 5-NITP and 5-PhITP that contain significant pi-electron density. As expected, the incorporation of either 5-NapITP or 5-AnITP opposite templating DNA is reduced and presumably reflects steric constraints imposed by the large size of the polycyclic aromatic moieties. Furthermore, 5-NapITP is a chain terminator of translesion DNA synthesis because the DNA polymerase is unable to extend beyond the incorporated non-natural nucleotide. In addition, idle turnover measurements confirm that 5-NapIMP is stably incorporated opposite damaged DNA, and this enhancement reflects the overall favorable incorporation kinetic parameters coupled with a reduction in excision by the exonuclease-proofreading activity of the enzyme. On the basis of these data, we provide a comprehensive assessment of the potential role of pi-electron surface area for nucleotide incorporation opposite templating and nontemplating DNA catalyzed by the bacteriophage T4 DNA polymerase.

Polymerases:

T4

Topics:

Nucleotide Analogs / Template Lesions, Nucleotide Incorporation

Status:

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Results:

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