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.


Enzymology:
 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.