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

Org Biomol Chem. 2007 Nov 21;5(22):3623-30. doi: 10.1039/b712480e. Epub 2007 Oct 12.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacteriophage T4 / enzymology
  • Catalysis
  • DNA / chemistry*
  • DNA / metabolism*
  • DNA Damage*
  • DNA-Directed DNA Polymerase / metabolism
  • Kinetics
  • Models, Chemical
  • Nucleotides / chemical synthesis
  • Nucleotides / chemistry
  • Nucleotides / metabolism*
  • Time Factors


  • Nucleotides
  • DNA
  • DNA-Directed DNA Polymerase