RESEARCH PLAN:
The aim of this KO4/RCDA application is to use a newly developed analytical technology to study metal-catalyzed OH. radical attack on DNA and proteins in minichromosomes and chromatin and on cell membranes. The newly developed assay makes use of a succinylated derivative of coumarin-3- carboxylic acid (hereafter referred to as SECCA) that is stably fluorescent when 7-hydroxylated during OH. attack. The resulting fluorescent derivative can be used in two ways: signal intensity following OH. damage gives a good idea of the amount of OH. damage in a given molecular compartment, while fluorescence polarization gives some idea of conformational changes that occur at damage sites. The plan is to place SECCA in histones (H1 or core), in DNA or in cell membranes, then measure fluorescence intensity and polarization anisotropy following Cu, Cr and Fe-catalyzed OH. damage. Assays for protein crosslinks and DNA damage (single- and double- strand breaks and 8-oxodG formation) will be run where appropriate on the same experimental samples.
