Year: 2008
Type: article
Abstract: BABHO DEVADOSSTranslesion 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 πelectron 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 5napthyl indole derivatives are incorporated 15-190-fold more efficiently than 5-NITP opposite the thymine dimer.In general, the differences in catalytic efficiency reflect xix 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.
Author: Babho Devadoss
Institution: Case Western Reserve University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: National Institutes of Health
OpenAlex ID: W2538506954