Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA.
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.
Vineyard
Diana
D
Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Zhang
Xuemei
X
Donnelly
Alison
A
Lee
Irene
I
Berdis
Anthony J
AJ
D017122:Bacteriophage T4 / Q000201:enzymology; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D009711:Nucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase