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Paper
Org. Biomol. Chem., 2007, 5, 3623 - 3630, DOI: 10.1039/b712480e
Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA
Diana Vineyard, Xuemei Zhang, Alison Donnelly, Irene Lee and Anthony J. Berdis
The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the
-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and -electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2
-deoxyriboside 5-triphosphate (5-CyITP), 5-ethyleneindolyl-2-deoxyriboside 5-triphosphate (5-EyITP), 5-methylindolyl-2-deoxyriboside 5-triphosphate (5-MeITP), and 5-ethylindolyl-2-deoxyriboside 5-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall -electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA 
100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.
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