The mechanisms governing the generation and distribution of metal-catalyzed OH. in the immediate vicinity of chromatin remain unclear due in part to lack of methods sufficiently specific to study these short-lived radicals. These researchers have developed an optical methodology to address this problem. Novel fluorescent probes for the detection of OH. have been developed by these researchers and these probes will be incorporated into specific sites of chromatin and genes of high metal affinity within chromatin to allow the simultaneous and independent detection of (i) metal catalyzed OH. formation; and (ii) chromatin conformation. Furthermore, chromatin-damage assays can be subsequently carried out on the same sample in order to relate cause (OH.) and effect (damage). This method will be used in isolated chromatin to (i) quantitate the accessibility of OH. catalyzed by copper, iron or chromium to specific chromatin regions, and (ii) correlate the optical information obtained in particular chromatin conformations with chromatin damage scored on the same samples in order to evaluate the distribution and significance of metal-catalyzed OH.. In a precisely characterized minichromosome system, the microdistribution of metal-catalyzed OH. within individual histones, DNA and the 5S rRNA gene and the binding site of the transcription factor TFIIIA on this gene will be examined. The project will also examine the feasibility of developing the method for use in living cells.
