Date of Award

Spring 1-1-2013

Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry & Biochemistry

First Advisor

Robert Kuchta

Second Advisor

Jennifer Kugel

Third Advisor

Charles McHenry

Fourth Advisor

Byron Purse

Fifth Advisor

Amy Palmer


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/O4 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/O4 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.