The central goal of this research is to delineate at the molecular level the structural features of eukaryotic chromatin which determine the susceptibility to damage by ionizing radiation and subsequent repair of the DNA housed therein. The experiments are designed to test the hypothesis (a) that DNA within relaxed chromatin is more sensitive to indirect radiation damage and (b) that repair involves a dynamic participation of a nuclear protein complex, perhaps the nuclear matrix. Mammalian cell lines have been selected because they permit the controlled modification of chromatin structure of defined genes or sequences and because appropriate genetic probes are available. Cell culture conditions will be modified to place the genes in either condensed or relaxed chromatin. The yield of radiation-induced single-strand breaks and DNA-protein cross-links will be determined, along with the kinetics of repair of those lesions, in the defined genes as compared to the bulk DNA. Damage will be determined by agarose electrophoresis, filter binding, and gel filtration chromatography, and the various genes will be probed by hybridization. The frequency of the particular genes in damaged vs. undamaged portions of the DNA will permit the calculation of the yield of lesions in the various chromatin sub-types. The protein complex which appears to be involved in DNA-protein cross-linking will be analyzed, and its protein components compared to those of the nuclear matrix. Acceptors of poly(ADP-ribose) polymerase will be identified and localized on active or inactive chromatin, or the nuclear matrix. Changes in the poly(ADP-ribosylation) of the acceptor proteins will be correlated with the repair of active and bulk chromatin. Until recently, most studies of ionizing radiation-induced damage in DNA considered the entire cellular DNA without regard for the nuclear environment. Modern biotechnology now permits the dissection of the DNA into regions which are more or less susceptible to radiation damage. The next advances in this area should come from the detailed analysis of the role of chromatin structure in determining the sensitivity of DNA to radiation damage and should assist in providing models of DNA damage and repair for application to clinical radiology.
Showing the most recent 10 out of 23 publications
