Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


case1222818842.pdf (4.07 MB)

ETD Abstract Container

Abstract Header

Probing the Base Stacking Contributions During Translesion DNA Synthesis

Devadoss, Babho
http://rave.ohiolink.edu/etdc/view?acc_num=case1222818842

Abstract Details

2008, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Translesion DNA synthesis is the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. This process can cause mutagenesis resulting in disease development. During this process, DNA polymerases recognize several biophysical features of the nucleotide during the incorporation opposite lesions to promote mutagenic events. Using a series of non-natural nucleotides as molecular probes, we have evaluated the contributions of base stacking, shape complementarity, and solvation energies toward nucleotide incorporation and exonuclease proofreading activity of gp43 DNA polymerase at an abasic site and thymine dimer lesions. In chapter 2, we used modified purine nucleotides to provide evidence that the π-electron system and hydrophobicity enhances the nucleotide incorporation at an abasic site, while the π-electron system alone facilitates the extension beyond a mispair. In chapter 3, we provided evidence that 5-NITP, a nucleotide that is preferentially incorporated opposite an abasic site, yields a ~2,600-fold lower catalytic efficiency opposite a thymine dimer compared to an abasic site and argues against the transient abasic site model proposed earlier with T7 DNA polymerase and Dpo4. In contrast, 5-cyclohexene, 5-phenyl, and 5-napthyl indole derivatives are incorporated 15-190-fold more efficiently than 5-NITP opposite the thymine dimer. In general, the differences in catalytic efficiency reflect perturbations in binding affinity in which nucleotides with larger π-electron surface area bind more favorably than those lacking π-electron density during incorporation at the thymine dimer. This is in contrast to an abasic site in which the binding affinity is independent of π-electron surface area. Kinetic evidence suggests that the incorporation of 5-PhITP and 5-CHITP opposite a thymine dimer is partially limited by the phosphoryl transfer step while incorporation opposite an abasic site is limited by the conformational change preceding phosphoryl transfer. In chapter 4, we used 5-NapITP as a fluorescent probe to demonstrate the existence of pre-exonuclease complex during exonuclease proofreading at either lesion. Our data suggest that the formation of the pre-exonuclease complex is driven by the base-stacking interactions with the lesions.
Irene Lee, PhD (Advisor)
James Burgess, PhD (Committee Chair)
Robert Salomon, PhD (Committee Member)
Mary Barkley, PhD (Committee Member)
Chris Dealwis, PhD (Committee Member)
230 p.
Biochemistry
Translesion DNA synthesis; DNA lesions; DNA polymerases

Recommended Citations

Citations

  • Devadoss, B. (2008). Probing the Base Stacking Contributions During Translesion DNA Synthesis [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1222818842

    APA Style (7th edition)

  • Devadoss, Babho. Probing the Base Stacking Contributions During Translesion DNA Synthesis. 2008. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1222818842.

    MLA Style (8th edition)

  • Devadoss, Babho. "Probing the Base Stacking Contributions During Translesion DNA Synthesis." Doctoral dissertation, Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1222818842

    Chicago Manual of Style (17th edition)

case1222818842
865
© 2008, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.