UVM Theses and Dissertations
Format:
Print
Author:
Zahn, Karl. E.
Dept./Program:
Cell and Molecular Biology Program
Year:
2013
Degree:
Ph. D.
Abstract:
Deducing the structure of the DNA double helix in 1953 implied the mode of its replication: Watson-Crick base pairing might instruct an enzyme, now known as the DNA polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. What has become increasingly clear in the last sixty years, however, is that all organisms conserve multiple DNA polymerases, specialized not only for DNA synthesis opposite adducted and oxidatively generated DNA bases, but also for unique biological roles such as somatic hypermutation or intricate DNA repair processes such as double-strand break repair.
This dissertation presents in six chapters the completed or currently underway structural investigations I have worked on, since joining the Doublie lab in 2006, aimed to further our understanding of the DNA polymerase's role in DNA metabolism. Chapter 1 provides a review of crystal structures describing DNA polymerares binding DNA lesions. Chapter 2 examines phosphonoformic acid (PFA, foscarnet), an antiviral drug that inhibits the human cytomegalovirus (HCMV) DNA polymerase (UL54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. By comparing two crystal structures that depict a RB69 gp43/UL54 chimeric DNA polymerase with and without PFA bound, we provide a model for the mechanism of UL54 stalling by PFA.
Chapter 3 investigates 5-hydroxycytosine (5-OHC), a stable oxidation product of cytosine associated with increased C[reaction direction]T transition mutations due to pairing with A. This study presents the first crystal structure of any DNA polymerase binding 5-OHC, showing that while dGMP is stabilized opposite 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template reminiscent of an abasic site. Chapter 4 reports four structures depicting several steps of translesion DNA synthesis site by RB69 gp43 exo- opposite an abasic site, employing a non natural purine triphosphate analog, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP.
Chapter 5 represents a work in progress, providing an overview of our continuing efforts to model human DNA polymerase [theta] (hPOLQ), a specialized polymerase overexpressed in breast cancer cells, among others. By solving a 3.9Å crystal structure, we have already obtained an atomic resolution glimpse of the polymerase active site. This dissertation concludes in chapter 6 with the strategies we will be applying in the near future to extend the resolution of our polymerase [theta] model and compose the next round of questions in the area of research.
This dissertation presents in six chapters the completed or currently underway structural investigations I have worked on, since joining the Doublie lab in 2006, aimed to further our understanding of the DNA polymerase's role in DNA metabolism. Chapter 1 provides a review of crystal structures describing DNA polymerares binding DNA lesions. Chapter 2 examines phosphonoformic acid (PFA, foscarnet), an antiviral drug that inhibits the human cytomegalovirus (HCMV) DNA polymerase (UL54) by mimicking the pyrophosphate leaving group of the nucleotide transfer reaction. By comparing two crystal structures that depict a RB69 gp43/UL54 chimeric DNA polymerase with and without PFA bound, we provide a model for the mechanism of UL54 stalling by PFA.
Chapter 3 investigates 5-hydroxycytosine (5-OHC), a stable oxidation product of cytosine associated with increased C[reaction direction]T transition mutations due to pairing with A. This study presents the first crystal structure of any DNA polymerase binding 5-OHC, showing that while dGMP is stabilized opposite 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template reminiscent of an abasic site. Chapter 4 reports four structures depicting several steps of translesion DNA synthesis site by RB69 gp43 exo- opposite an abasic site, employing a non natural purine triphosphate analog, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP.
Chapter 5 represents a work in progress, providing an overview of our continuing efforts to model human DNA polymerase [theta] (hPOLQ), a specialized polymerase overexpressed in breast cancer cells, among others. By solving a 3.9Å crystal structure, we have already obtained an atomic resolution glimpse of the polymerase active site. This dissertation concludes in chapter 6 with the strategies we will be applying in the near future to extend the resolution of our polymerase [theta] model and compose the next round of questions in the area of research.