Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA.

Abstract:

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 pi-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 pi-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 pi-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA approximately 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.

Polymerases:

T4

Topics:

Nucleotide Analogs / Template Lesions, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

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