The fundamental mechanisms underlying the generation of radiation lesions in cellular chromatin remain unclear, due in part to lack of experimental methods sufficiently specific to probe existing hypotheses. It has been postulated that generation of unrepairable DNA damage is related to the distribution of initial chromatin lesions, a combined result of the conformation of specific chromatin sites and the frequency of hydroxyl radicals (OH) and direct ionizations. Current chromatin damage assays are not site-specific and they only score the net result of many processes. We have developed a novel technology designed to simultaneously record in the same sample (a) OH distribution within specific sites on chromatin, (b) conformation of chromatin within these sites, and (c) chromatin damage as determined by any currently used assay. By correlating the information obtained, our studies aim to understand fundamental processes underlying the generation of deleterious radiation damage. Novel fluorescent probes are incorporated into preferred sites within chromatin and upon irradiation allow the collection of two independent fluorescent signals, one that records radiation-induced OH and another that records conformational changes at these sites. Since the two optical signals can be detected immediately following OH attack, the present technology allow sensitive, real-time registration of OH during active chromatin conformational changes. Optical methods will be used to quantitate the accessibility of radiation-induced OH to chromatin components (specific histones, DNA sites). The site-specific information obtained in particular chromatin conformations will be correlated to overall damage scored on the same samples (DNA strand breaks and DNA- histone crosslinks). Since the distribution of initial chromatin damage and its ability to repair depend on LET, OH will be generated by gamma rays (low LET), alpha particles (high LET), and Auger electrons (positionally dependent LET) to elucidate mechanisms underlying the generation of unrepairable damage. This knowledge would greatly augment the development of strategies for the beneficial modification of therapeutic uses of radiation and the evaluation of potential risks associated with its diagnostic use as well as the assessment of its environmental hazards (e.g. exposure to radon).
