Physics & Chemistry of DNA Damage Produced by Ionizing Radiation
Crystal structure of d(CCAGTACTGG)2 viewed parallel to the helix axis (left) and perpendicular to the helix axis (right). Structure solved and published by C. L. Kielkopf, S. Ding, P. Kuhn, and D. C. Rees, Conformational Flexibility of B-DNA at 0.74 Å Resolution: d(CCAGTACTGG)2. J. Mol. Biol. 296, 787-801 (2000).
The long-term objective is to determine the mechanisms by which ionizing radiation, through direct effects, alters DNA structure. Direct effects accounts for 30-50% of the in vivo DNA damage for low LET (linear energy transfer) radiations. At high LET, direct effects account for ˜80% of the damage. Knowledge of the mechanism by which ionizing radiation produces damage in DNA is critical in determining the risks, such as induction of cancer or leukemia, due to low dose and low dose rates of radiation. Achievement of the stated objective will be a major benefit in risk assessment and disease treatment.
The specific aims are:
- Understanding of Hole and Excess-Electron Trapping and Detrapping by DNA
- Reactions that Lead to the Formation of Radicals that are Trapped in DNA
- Correlate Stable End Products with Their Free Radical Precursors
- Detect Multiply Damaged Sites and Determine Their Yields
- Size Based Filtration of Oligodeoxynucleotides
The approach is to use electron paramagnetic resonance (EPR) spectroscopy to study free radical intermediates formed in oligodeoxynucleotides and DNA. Oligodeoxynucleotides are studied in crystalline form and plasmid DNA is studied in the form of films. EPR measurements are made at 4K to maximize detection sensitivity. X-irradiation (70 kV) can be delivered at 4K, 120K, 240K, RT in order to ascertain the effect of radiation temperature. Stable end products are analyzed using the same samples as employed for EPR. Strand breaks are identified using high performance liquid chromatography. Base damage is determined using gas chromatography/mass spectrometry.
Central to our design is the use of DNA samples (primarily oligodeoxynucleotides) that are structurally well defined. The key samples are crystalline. By employing crystals of DNA, for which the structure has been solved by x-ray diffraction, we maximize our knowledge regarding base sequence, DNA conformation, hydration state, counter ions, packing, and purity. These qualities are illustrated by the example shown above. The oligomer sequence is d(CpCpApGpTpApCpTpGpG); the conformation is B-DNA with a rise of 3.3 Å per residue; there are 13.1 H2O per residue (50.1 % of the crystal is solvent); there are 8 Ca++ per duplex (given that the duplex has no terminal phosphates, there are 8 PO4/duplex.); and the duplexes are packed end-to-end forming continuous cylinders that run parallel to one another.
Three features are particularly noteworthy. First, base stacking is seamless at the point of abutment between two decamers; with regard to base stacking, therefore, this DNA has the properties of high molecular weight DNA (104-106 bp). The base-stacking length depends on crystal dimensions, typically 0.01-1.0 mm. By choosing crystals of other oligomers, the length of base stacking is readily limited to the length of one oligomer. Second, in this example all the bases lie in the same plane, optimizing the information that can be obtained by EPR. And third, solvent channels run continuously through the crystals, making it possible to diffuse small molecules such as O2 through the crystal.
- Identification of the C4'-Oxidized Abasic Site as the Most Abundant 2-Deoxyribose Lesion in Radiation-Damaged DNA Using a Novel HPLC-Based Approach. Radiat Res. In press. (2014 Jan 10).
- Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies. Acta Crystallogr D Biol Crystallogr. 69, 2381-94. (2013 Dec 01).
- DNA damage by the direct effect of ionizing radiation: products produced by two sequential one-electron oxidations. J Phys Chem A. 117, 12608-15. (2013 Nov 27).