Lund, Travis John.
Asymmetric Recognition of Nucleobase Features by DNA Polymerases.
This work describes an investigation into the recognition of nucleobase features by several DNA polymerases. I used of a series of pyrimidine analogues modified at O2
, N-3, and N4
to determine how the Klenow fragment of DNA polymerase I, an A family polymerase, and two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether or not to polymerize pyrimidine dNTPs. Removal of these heteroatoms generally impaired polymerization, with the effects varying from mild to severe. Removing O2
of a pyrimidine dNTP vastly decreased incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O2
from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N4
greatly impaired polymerization, both of the resulting dNTP analogues as well as polymerization of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Removing O2
from a pyrimidine at the primer 3'-terminus also prohibited extension of the primer. Importantly, these studies indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. I also describe initial work on the synthesis of a novel dibasic analogue incorporating chemical features whose importance has been demonstrated in this work. These features include the presence of minor groove hydrogen bond acceptors to facilitate extension past the analogue upon its incorporation, as well as the Watson-Crick hydrogen bonding groups of an A:T pair, since we have seen that the removal or modification of these groups has unpredictable, but often detrimental, effects upon efficient nucleobase incorporation by polymerases.
Advisors/Committee Members: Robert Kuchta, Jennifer Kugel, Charles McHenry, Byron Purse, Amy Palmer.
to Zotero / EndNote / Reference
APA (6th Edition):
Lund, T. J. (2013). Asymmetric Recognition of Nucleobase Features by DNA Polymerases. (Doctoral Dissertation). University of Colorado. Retrieved from https://scholar.colorado.edu/chem_gradetds/75
Chicago Manual of Style (16th Edition):
Lund, Travis John. “Asymmetric Recognition of Nucleobase Features by DNA Polymerases.” 2013. Doctoral Dissertation, University of Colorado. Accessed April 01, 2020.
MLA Handbook (7th Edition):
Lund, Travis John. “Asymmetric Recognition of Nucleobase Features by DNA Polymerases.” 2013. Web. 01 Apr 2020.
Lund TJ. Asymmetric Recognition of Nucleobase Features by DNA Polymerases. [Internet] [Doctoral dissertation]. University of Colorado; 2013. [cited 2020 Apr 01].
Available from: https://scholar.colorado.edu/chem_gradetds/75.
Council of Science Editors:
Lund TJ. Asymmetric Recognition of Nucleobase Features by DNA Polymerases. [Doctoral Dissertation]. University of Colorado; 2013. Available from: https://scholar.colorado.edu/chem_gradetds/75