A unique property of ionizing radiation and radiomimetic chemotherapeutic drugs is the generation of clustered DNA damage, this is two or more DNA lesions (oxidized bases, modified sugars, SSB, and DSB) located within a single turn of the DNA helix. It has been known for some time that the number of DSB correlates directly with the kill effects of ionizing radiation. In addition to DSB, multiply damaged sites (MDS) composed of base and/or sugar damage and/or SSB, are readily produced in the cell after low doses of ionizing radiation, and that they make up to 80% of the total clustered damage. It has been shown recently that, attempts to repair these MDS can produce different outcomes, depending on the type of damages, their separation, and relative orientation. DNA incision studies using purified DNA glycosylases or nuclear cell extracts showed that some MDS can be cleaved readily generating toxic DSB, while others are incised very poorly, persisting in the cell for longer periods of time. MDS made of identical lesions can be processed differently depending on damage separation and relative orientation. At the present time, the structural basis that explains this property is almost non-existent. In this application we propose to determine the solution structure of different types of clustered bistrand lesions, to correlate the structures with their recognition by purified DNA glycosylases, and to study their processing by cellular extracts. We will use high-resolution NMR spectroscopy in combination with restrained molecular dynamics to determine three-dimensional structures of DNA duplexes containing clustered bistrand lesions formed by a combination of damage bases (8-oxoG, DHT), abasic sites, or strand breaks, varying inter-lesion separation and orientation. We will assay cleavage of these lesions using purified DNA glycosylases to determine their recognition and processing by BER enzymes. We will assay the repair potential of the above-mentioned MDS using eukaryotic nuclear cell extracts to establish the extent and hierarchy of repair. Completion of this proposal will establish direct correlations between the solution structure of clustered DNA lesions and some of their biological properties, and will help to understand the molecular mechanisms of toxicity and mutagenicity of ionizing radiation, a knowledge with potential application for the design of novel chemotherapeutic drugs.
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