It is generally accepted that damage to DNA is the cause of cell death by ionizing radiation. A lesion in the DNA which has particular importance in this respect is the double strand break (DSB). From considerations of the mechanisms of production of DSB it is reasoned that locally multiply damaged sites (LMDS), which include base damages as well as strand breaks, are formed by similar mechanisms. These LMDS could also be biologically significant for cell survival in the presence of efficient DSB repair. Similar arguments can be made for the significance of these lesions for other cellular effects of irradiation - mutation and transformation. There have been no previous studies of the structures of these types of lesion either in cellular DNA or in model systems. Here we propose to study the structures of the LMDS and DSB produced as it would be in a cell. A model system, the SV40 minichromosome irradiated in a simulated cellular environment, will be used for the studies, and the specific benefits of this approach are discussed. The model system has been characterized and validated as representative of the DNA in a mammalian cell and a good understanding of the parameters of radiation damage to these molecules has been attained.
The Specific Aims of the proposed work are to describe the details of the structures of the biologically significant LMDS with a view to considering a cell's ability to repair them. There are three major Specific Aims in the project and they are: 1. Determine the relative yields of base damage to strand breaks within the LMDS. 2. Determine the spectrum of sizes of the LMDS in terms of numbers of base pairs. 3. Determine the average number of individually damaged sites per LMDS. All of the information gained from these studies will be significant in considering a cell's response to ionizing radiation. A series of novel assays specific for these purposes has been designed with these goals in mind. Several different radiation sources will be employed for the completion of the studies including gamma-rays, soft X-rays and high LET particles. Additional minor studies with this system include: Confirmation of the mathematical model for the production of DSB by coincident SSB; determination of DNA-histone cross-links; development of a general base damage assay; and, development of general method for assaying LMDS.
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