Document Type
Article
Publication Date
8-13-2007
Publication Title
ChemBioChem
Abstract
The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the “blunt-end” of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive π-electron surface areas are efficiently utilized by the T4 DNA polymerase. These analogues possess high binding affinities that are remarkably similar to those measured during incorporation opposite an abasic site. In contrast, the kpol values are significantly lower during blunt-end extension when compared to incorporation opposite an abasic site. These kinetic differences suggest that the single-stranded region of the DNA template plays an important role during polymerization through stacking interactions with downstream bases, interactions with key amino acid residues, or both. In addition, we demonstrate that terminal deoxynucleotide transferase, a template-independent enzyme, can efficiently incorporate many of these non-natural nucleotides. However, that this unique polymerase cannot extend large, bulky non-natural nucleotides suggests that elongation is limited by steric constraints imposed by structural features present within the polymerase. Regardless, the kinetic data obtained from using either DNA polymerase indicate that template-independent synthesis can occur without the contributions of hydrogen-bonding interactions and suggest that π-electron interactions play an important role in polymerization efficiency when templating information is not present.
Recommended Citation
Berdis, Anthony J. and McCutcheon, David, "The Use of Non-natural Nucleotides to Probe Template-Independent DNA Synthesis" (2007). Chemistry Faculty Publications. 223.
https://engagedscholarship.csuohio.edu/scichem_facpub/223
DOI
10.1002/cbic.200700096
Version
Postprint
Publisher's Statement
This is the accepted version of the following article: Berdis, A. J.; McCutcheon, D. The Use of Non-natural Nucleotides to Probe Template-Independent DNA Synthesis. ChemBioChem 2007, 8, 1399-1408., which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/cbic.200700096/abstract
Volume
8
Issue
12
Comments
This research was supported by funding from the National Institutes of Health to A.J.B. (CA118408).