The long-term objective of this proposed research program is to (i) measure the types, yields and spatial distribution of the biologically relevant DNA lesions produced by the direct effect of ionizing radiation, (ii) determine the chemical mechanisms by which these DNA lesions are formed, and (iii) evaluate how molecules such as histones and other DNA binding proteins modulate the yield and distribution of these DNA lesions. Achievement of these objectives should make it possible to predict the composition and spatial distribution of the DNA lesions within biologically important clusters. This predictive capability is central to making risk/benefit decisions at both low dose rates and low doses of radiation and to constructing biologically relevant models of clustered DNA damage to be used by DNA enzymologist, biochemists and biophysicists in their DNA repair/misrepair studies.
The specific aims are: 1) to determine the yields of specific sugar and base damage end-products produced by the direct effect as a function of radiation dose, and elucidate the reaction mechanisms giving rise to them, 2) to determine the yield and composition of DNA damage within clusters produced by the direct effect, and 3) to determine how histones and other protein DNA binding complexes modify the yield and composition of clustered DNA damage produced by the direct effect. Damage to DNA by the direct effect occurs via two routes. One is by direct ionization of the DNA. The other is by ionization of that portion of the solvent shell that is tightly bound to the DNA and rapid transfer of that damage to the DNA. Because of these properties, information on the direct effect is optimized by studying DNA in the solid state. The DNA samples, for this proposed research program, will be prepared in the form of crystals and films. Using crystals of known structure, we maximize our knowledge of relevant parameters: base sequence, conformation, hydration state, counter ions, packing, and purity. Using films we are able to vary parameters such as base sequence and degree of hydration. Unstable free radical intermediates will be studied by electron paramagnetic resonance (EPR) spectroscopy. Stable end products will be quantified by high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS). Stable end products will be analyzed using the same samples as those studied by EPR. Key elements in our experimental design are the use of structurally well defined DNA samples, making it possible to extend our knowledge of free radical reactions to understand the mechanisms of end product formation at low dose. By achieving the goals set down in this proposal, we will improve our ability to make risk/benefit decisions at low dose rates and low doses of radiation. Further, it provides the information needed to construct biologically relevant models of clustered DNA damage that can be used in studies of DNA repair and misrepair. The long-term objective of this proposed research program is to, (i) measure the types, yields and spatial distribution of the biologically relevant DNA lesions produced by the direct effect of ionizing radiation, (ii) determine the chemical mechanism(s) by which these DNA lesions are formed, and (iii) evaluate how molecules such as histones and other DNA binding proteins modulate the yield and distribution of these DNA lesions. Achievement of these objectives should make it possible to predict the composition and spatial distribution of the DNA lesions within biologically important clusters. This predictive capability is central to making risk/benefit decisions at both low dose rates and low doses of radiation and to constructing biologically relevant models of clustered DNA damage to be used by DNA enzymologist, biochemists and biophysicists in their DNA repair/misrepair studies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA032546-37
Application #
8215661
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Bernhard, Eric J
Project Start
1982-02-01
Project End
2013-09-30
Budget Start
2012-02-01
Budget End
2013-09-30
Support Year
37
Fiscal Year
2012
Total Cost
$447,791
Indirect Cost
$154,674
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Roginskaya, Marina; Mohseni, Reza; Moore, Terence J et al. (2014) 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 181:131-7
Peoples, Anita R; Lee, Jane; Weinfeld, Michael et al. (2012) Yields of damage to C4' deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation. Nucleic Acids Res 40:6060-9
Black, Paul J; Bernhard, William A (2011) EPR detection of an electron scavenging contaminant in irradiated deoxyoligonucleotides: one-electron reduced benzoyl. J Phys Chem B 115:8009-13
Sharma, Kiran K K; Swarts, Steven G; Bernhard, William A (2011) Mechanisms of direct radiation damage to DNA: the effect of base sequence on base end products. J Phys Chem B 115:4843-55
Price, Charles S; Razskazovskiy, Yuriy; Bernhard, William A (2010) Factors affecting the yields of C1' and C5' oxidation products in radiation-damaged DNA: the indirect effect. Radiat Res 174:645-9
Peoples, Anita R; Mercer, Kermit R; Bernhard, William A (2010) What fraction of DNA double-strand breaks produced by the direct effect is accounted for by radical pairs? J Phys Chem B 114:9283-8
Sharma, Kiran K K; Tyagi, Rahul; Purkayastha, Shubhadeep et al. (2010) One-electron oxidation of DNA by ionizing radiation: competition between base-to-base hole-transfer and hole-trapping. J Phys Chem B 114:7672-80
Sharma, Kiran Kumar K; Bernhard, William A (2009) Direct damage to the backbone of DNA oligomers is influenced by the OH moiety at strand ends, by the type of base, and by context. J Phys Chem B 113:12839-43
Sharma, Kiran K; Razskazovskiy, Yuriy; Purkayastha, Shubhadeep et al. (2009) Mechanisms of strand break formation in DNA due to the direct effect of ionizing radiation: the dependency of free base release on the length of alternating CG oligodeoxynucleotides. J Phys Chem B 113:8183-91
Sharma, Kiran Kumar K; Milligan, Jamie R; Bernhard, William A (2008) Multiplicity of DNA single-strand breaks produced in pUC18 exposed to the direct effects of ionizing radiation. Radiat Res 170:156-62

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