My Research Interests
Free Radical Chemistry:
The focus of my research efforts is on elucidating the mechanisms by which DNA is damaged by ionizing radiation and oxidative stress. Radiation induced DNA damage results from either direct ionization of the DNA macromolecule or from free radicals generated within the surrounding environment. Of particular importance in my research is assessing the influence that water content has on the formation of radiation induced DNA damage products.
Eukaryotic cells contain double stranded DNA bearing about 6x109 nucleotide base pairs. A polymer of this size if fully extended would be about 60 cm in length. In order to fit inside the cell nucleus, which is about 50mm in diameter, DNA must undergo a 100,000 fold reduction in apparent length. Nature solves this dilemma in eukaryotic cells by wrapping genomic DNA around the histone proteins, forming chromatin. The compaction of DNA into the small volume of the nucleus, as well as association of DNA with histones and other nuclear proteins(enzymes, lamins, microfilaments, etc.) results in the exclusion of bulk water near the DNA. The DNA in chromatin exists as an intermediate between the solution state and the solid state.
Classically it has been thought that irradiation of the
bulk water surrounding the DNA produces free radicals( e.g. the hydroxyl radical
and the aqueous electron) that can diffuse to and react with DNA. In my
work with Dr. Steven Swarts( Bowman Gray School of Medicine, Wake Forest
University, Winston-Salem NC) we have been able to establish that the water
molecules located closest to the DNA are qualitatively different from bulk
water. The irradiation of the water molecules tightly bound to the
DNA, within the first hydration layer, will damage DNA through a charge transfer
mechanism. By this mechanism, electron deficient "holes" and
electrons from the first hydration layer migrate to the DNA. The damage
from charge transfer processes is similar to damage expected from direct
ionization of the DNA(e.g. base release, dihydrothymine, dihydrocytosine), but
different from that expected from bulk water radical attack on DNA(e.g.strand
breaks, thymine glycol). This is significant because the tightly bound
water molecules, which have largely been overlooked, constitute
approximately 50% of the water that surrounds DNA in a cellular environment,
with the other half constituting bulk water. Further characterization of
the tightly bound water molecules and their role in radiation induced DNA
damage is necessary. I plan to extend this work to consider the
effects of irradiation of DNA in the presence of free radical scavengers
such as thiols, and in the presence of nucleoproteins.
The measurements described above are obtained from reverse phase high performance liquid chromatography(HPLC) and gas chromatography/ mass spectrometry (GC/MS) techniques that have been developed and optimized to quantify irradiated DNA end products. Novel product identification and characterization are done using the GC/MS in total ion mode, or using proton 1D nuclear magnetic resonance (NMR) spectroscopy.
I am interested in elucidating the structure/ activity relationships between DNA processing enzymes and various damaged DNA substrates, with a goal of developing sensitive techniques for detection of DNA damage in vivo.
It has been shown that for equal exposure to ionizing radiation, the yield of DNA base lesions in vitro is at least two orders of magnitude higher than what is observed in irradiated cells . Similarly, chromatin has been shown to be 100 times less susceptible to strand breaks and base damage by ionizing radiation and oxidative stress compared to histone depleted DNA . The protective effect of histone proteins, compact higher order chromatin structures, and cellular repair processes, are likely responsible for the empirically observed differences in damage susceptibilities. This emphasizes the need for development of highly sensitive assay techniques in order to detect irradiation or oxidatively induced damage within a cellular system.
As part of my graduate work with Dr. Harold Box (State University of New York at Buffalo/ Roswell Park Memorial Cancer Institute) I characterized the structure / activity relationships between radiation damaged DNA and endonuclease P1 from Penecillium citrinum.
Nuclease P1 is an endonuclease which functions as a phosphodiesterase, cleaving the bond between the 3’-hydroxyl and 5’-phosphoryl group of adjacent nucleosides. Nuclease P1 is capable of hydrolyzing single stranded DNA and RNA completely to the level of mononucleoside 5’-monophosphates. During my studies I had found that the efficiency of nuclease P1 in hydrolyzing the phosphodiester bonds of substrates damaged by ionizing radiation or oxidative stress may be significantly altered by modifications to the 5’ terminal base. The hydrolytic activity has been shown to be reduced by several orders of magnitude with the loss of base aromaticity resulting from saturation of the 5-6 double bond of thymine when present on the 5’ terminus. Several DNA lesions were determined to be slowly hydrolyzed or completely refractory to hydrolysis by nuclease P1.
DNA is hydrolyzed completely to the level of mononucleoside 5’-monophosphates by nuclease P1. Damaged DNA species that were refractory to hydrolysis were isolated as dinucleoside monophosphates by high performance liquid chromatography(HPLC). A probabilistic model was derived to calculate the hydrolytic course of a DNA polymer by nuclease P1 in order to isolate slowly hydrolyzed species. As a result of this work, a sensitive 32P postlabeling assay has been developed for the detection of the formamido remnant of pyrimidine bases, a refractory lesion to nP1. The technique has successfully been applied in quantifying this lesion in irradiated keratinocytes. Currently I am extending this technique to assay for DNA base lesions which are slowly hydrolyzed by nP1. I plan to extend the technique to detect abasic sites within a DNA polymer. I plan to further develop this method of detection of radiation and/or oxidatively induced DNA damage using different enzyme systems, and hope to apply the techniques to detect and characterize DNA damage in vivo.