Summary of My Research at The University of Rochester

In my graduate research, I investigated the early radiation chemistry of DNA. Specifically, I was interested in radiation induced free radical damage in DNA exposed to high energy radiation.      High energy radiation induces ionization events, which generate holes (via electron abstraction) and thermalized electrons. Molecular scavenging of electrons and holes can produce free radicals. Free radicals are reactive species possessing an unpaired electron(s); they are the early precursors to stable damage in DNA.      The role of free radicals in disease pathogenesis is a topic of recent interest. Radiation induced damage is unique (versus chemically induced red-ox damage) in that the damage is formed in molecular clusters, feasibly augmenting the extent and/or nature of the damage.      In our work, DNA samples were irradiated with X-rays, generated by a Varian/Eimac OEG-76 X-ray tube. Electron paramagnetic resonance (EPR) spectroscopy was used to study the free radical intermediates. Free radicals are relatively unstable at ambient temperatures. Hence, a Janis cryostat was used to cool the samples to 4 K.      Electron and hole migration is one of the underlying processes affecting the free radical chemistry in DNA. For example, migration can proceed along the base stack in DNA. Migration is terminated by radical trapping events and radical combination events. This migration influences the distribution and extent of radiation induced damage.      Efficient migration of electrons and holes over short distances (£ 2 base pairs) in DNA was evident from our structural EPR studies of irradiated anthracycline- intercalated DNA oligomers. A mechanism of electron and hole transfer, from the DNA solvation layer to the DNA molecule, was supported by quantitative measures of free radical yields in variably hydrated DNA samples.      Migration to the DNA bases is energetically favorable, as the bases are readily oxidized and reduced. Hole migration to the sugar-phosphate backbone is also possible, if rapid deprotonation follows hole capture. Hole scavenging by a minor-groove associated amino-sugar was observed, supporting the notion that sugar radicals can be trapped in irradiated DNA.      The yield of free radicals is determined by the extent of radical trapping relative to radical combination. DNA packing (the molecular arrangement of DNA and water molecules) is one variable that appears to influence these processes. In DNA with relatively close intermolecular spacing, radicals are more prone to be scavenged onto separate DNA molecules; the greater dielectric shielding in tightly compacted DNA promotes radical trapping. DNA hydration waters also act to stabilize free radical damage on DNA, most likely as a result of proton transfer reactions. Hence, both the physical hydration and the packing state of DNA influence quantitative damage.      Electrons and holes that encounter DNA are readily trapped; hence long-distance migration along the base stack does not appear to be facile (with long distance migration, electrons and holes would more readily annihilate each other, resulting in low free radical yields) . This is particularly evident in the continuously stacked DNA oligomer crystal, d(CCAACGTTGG), in which free radical yields are relatively high. Relatively rapid proton transfer reactions are thought to stabilize radical damage, thus hindering migration.      The recently hypothesized assertion that DNA behaves as a molecular wire is inconsistent with our free radical yield data; furthermore, it contradicts the commonly accepted observation that DNA is susceptible to radiation damage.           Bill Bernhard (Professor of Biochemistry and Biophysics), was my graduate/research advisor. Kermit Mercer (Associate in Biochemistry and Biophysics), an equipment engineer (with too many stories), was also an invaluable asset in my training.      The Department of Biochemistry and Biophysics page, and the Biophysics & Structural Biology Cluster page, provide further information about Biophysics at the University of Rochester.

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