UVM Theses and Dissertations
Format:
Print
Author:
Dunn, Andrew R.
Dept./Program:
Cell and Molecular Biology Program
Year:
2012
Degree:
Ph. D.
Abstract:
Steady state measurements estimate that nearly 1 in every 10⁵ normal bases has become damaged through an oxidation event. Reactive oxygen species (ROS) generated from normal cellular respiration can be harmful to DNA causing base damages that produce potentially detrimental mutations when left unrepaired. To combat the constant onslaught of damage production, cells have evolved a network of safeguards to preserve genomic integrity. The repair mechanism responsible for the removal of the majority of oxidative base damages is the base excision repair (BER) pathway.
The critical first step in the BER pathway is accomplished by DNA N-glycosylases, which locate and remove single damaged bases. Escherichia coli endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families, the helix-hairpin-helix (HhH) superfamily and the Fpg/Nei family, respectively. Using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with elongated [lamda]DNA molecules, we have shown that members of the HhH and the Fpg/Nei families have similar one-dimensional search mechanisms. In addition, the mean diffusion constants for E. coli Fpg, Nei and Nth were in keeping with theoretical approximations for rotational diffusion along the DNA. Interestingly, the characteristic motion was described as a continuum of motion ranging from slow, subdiffusive to faster, unrestricted diffusion.
Driven by these observations and crystal structures showing that glycosylases may locate damages through insertion of a 'wedge' residue between two sets of base pairs, we found that mutant E. coli, Fpg F111A, Nei Y72A and Nth L8IA, which lack the wedge residue, no longer displayed slow, sub-diffusive motion compared to wild type. These data suggest that damage location may be accomplished through testing the relative strength and flexibility of base pairs by temporally elongating Watson-Crick hydrogen bonds upon wedge insertion. If a damaged base is present and does not make strong hydrogen bonds it may become destabilized by the presence ofthe wedge residue and be extruded into the active site where further interrogation can occur.
To further testthis hypothesis we generated [lamda]-DNA molecules that contained 8-oxo-7,8-dihydroguanine (8-oxoG) and 5,6 dihydroxy-5,6 dihydroxy-5,6 dihydrothymine (thymine glycol (Tg)) damages for Fpg and NeilNth, respectively. We observed that as the concentration of damages increases the binding lifetimes for wild type Fpg, Nei and Nth also increases. However, the wedge mutants do not show any correlation between damage concentration and binding lifetime. In addition, the mean diffusion constants of wild type Fpg, Nei and Nth decrease with increasing concentration of amages, whereas those of the wedge mutants do not. These data suggest that the glycosylase search mechanism may occur through intrahelical interrogation and provides the most parsimonious explanation of how this thermally driven search is capable of locating damages while one-dimensionally moving along DNA at diffusion limits.
The critical first step in the BER pathway is accomplished by DNA N-glycosylases, which locate and remove single damaged bases. Escherichia coli endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families, the helix-hairpin-helix (HhH) superfamily and the Fpg/Nei family, respectively. Using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with elongated [lamda]DNA molecules, we have shown that members of the HhH and the Fpg/Nei families have similar one-dimensional search mechanisms. In addition, the mean diffusion constants for E. coli Fpg, Nei and Nth were in keeping with theoretical approximations for rotational diffusion along the DNA. Interestingly, the characteristic motion was described as a continuum of motion ranging from slow, subdiffusive to faster, unrestricted diffusion.
Driven by these observations and crystal structures showing that glycosylases may locate damages through insertion of a 'wedge' residue between two sets of base pairs, we found that mutant E. coli, Fpg F111A, Nei Y72A and Nth L8IA, which lack the wedge residue, no longer displayed slow, sub-diffusive motion compared to wild type. These data suggest that damage location may be accomplished through testing the relative strength and flexibility of base pairs by temporally elongating Watson-Crick hydrogen bonds upon wedge insertion. If a damaged base is present and does not make strong hydrogen bonds it may become destabilized by the presence ofthe wedge residue and be extruded into the active site where further interrogation can occur.
To further testthis hypothesis we generated [lamda]-DNA molecules that contained 8-oxo-7,8-dihydroguanine (8-oxoG) and 5,6 dihydroxy-5,6 dihydroxy-5,6 dihydrothymine (thymine glycol (Tg)) damages for Fpg and NeilNth, respectively. We observed that as the concentration of damages increases the binding lifetimes for wild type Fpg, Nei and Nth also increases. However, the wedge mutants do not show any correlation between damage concentration and binding lifetime. In addition, the mean diffusion constants of wild type Fpg, Nei and Nth decrease with increasing concentration of amages, whereas those of the wedge mutants do not. These data suggest that the glycosylase search mechanism may occur through intrahelical interrogation and provides the most parsimonious explanation of how this thermally driven search is capable of locating damages while one-dimensionally moving along DNA at diffusion limits.