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Investigation of Noncanonical DNA Polymerases and Their Mechanisms

Fowler, Jason David

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2009, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.

DNA polymerases have evolved complex biological functions to balance the need for faithful DNA replication with the need for subtle genetic flexibility in the form of random mutations that are necessary for sustaining life in an ever-changing environment. A more fundamental understanding of the diverse mechanisms that lie at the heart of DNA polymerases is the ultimate goal of the ongoing research presented here.

Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine (dFdC), is a drug approved for use against various solid tumors. Clinically, this moderately toxic nucleoside analog causes side effects which closely mimic symptoms of mitochondrial dysfunction, although there is no direct evidence to show that gemcitabine interferes with mitochondrial DNA replication catalyzed by human DNA polymerase gamma. Here we employed pre-steady state kinetic methods to directly investigate the incorporation of the 5'-triphosphorylated form of gemcitabine (dFdCTP), excision of the corresponding incorporated monophosphorylated form (dFdCMP), and bypass of template base dFdC catalyzed by human DNA polymerase gamma. Opposite template base dG, dFdCTP was incorporated with a 432-fold lower efficiency than dCTP. Although dFdC is not a chain terminator, the incorporated dFdCMP decreased the incorporation efficiency of the next two correct nucleotides by 214- and seven-fold, respectively. Moreover, the primer 3'-dFdCMP was excised with a 50-fold slower rate than the matched 3'-dCMP. When dFdC was encountered as a template base, DNA polymerase gamma paused at the lesion and one downstream position but eventually elongated the primer to full-length product. These pauses were because of a 1,000-fold decrease in nucleotide incorporation efficiency. Interestingly, the polymerase fidelity at these pause sites decreased by two orders of magnitude. Thus, our pre-steady state kinetic studies provide direct evidence demonstrating the inhibitory effect of gemcitabine on the activity of human mitochondrial DNA polymerase.

Crystallographic studies of the truncated C-terminal DNA polymerase-beta-like domain of human DNA polymerase lambda (tPol lambda) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with replicative DNA polymerases and DNA polymerase beta. To examine solution-phase protein conformational changes in full length DNA polymerase lambda (fpol lambda), which contains two additional domains at its N-terminus, we used a mass spectrometry-based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPollambda versus the binary complex of enzyme*gapped DNA and the ternary complex of enzyme*gapped DNA*dNTP (2'-deoxynucleotide triphosphate). Results from these experiments suggest that fPollambda does not undergo major conformational changes during catalysis in the solution phase. Furthermore, mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide. Site-directed mutagenesis and pre-steady-state kinetic studies confirmed the importance of R386 for the enzyme activity and indicate a key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and are important for catalytic functions of other DNA polymerases.

Zucai Suo, PhD (Advisor)
Venkat Gopalan, PhD (Committee Member)
Mamuka Kvaratskhelia, PhD (Committee Member)
Chenglong Li, PhD (Committee Member)
201 p.

Recommended Citations

Citations

  • Fowler, J. D. (2009). Investigation of Noncanonical DNA Polymerases and Their Mechanisms [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1250701062

    APA Style (7th edition)

  • Fowler, Jason. Investigation of Noncanonical DNA Polymerases and Their Mechanisms. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1250701062.

    MLA Style (8th edition)

  • Fowler, Jason. "Investigation of Noncanonical DNA Polymerases and Their Mechanisms." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1250701062

    Chicago Manual of Style (17th edition)