The goal of this proposal is to elucidate the molecular mechanisms in human cells responsible for repair of a specific type of lesion introduced into DNA by ultraviolet (UV) radiation, the pyrimidine dimer. Of the various DNA lesions produced by UV radiation in sunlight, the pyrimidine dimer is especially important in the etiology of sun- induced skin cancer as well as a number of other pathological effects. It is also among the DNA adducts most increased by depletion of the atmospheric ozone layer. We have isolated a DNA endonuclease complex from normal human cell chromatin which is composed of both a DNA endonuclease with specificity for pyrimidine dimers and a protein necessary for interaction of this endonuclease with damaged nucleosomal DNA. This complex scans the DNA molecule so as to identify damaged sites in a processive manner. We have shown that, in cells derived from patients with the sun-sensitive, cancer prone, DNA repair-deficient genetic disease, xeroderma pigmentosum, complementation group A (XPA), this endonuclease complex both is deficient in ability to incise damaged nucleosomal DNA and is unable to scan the damaged DNA molecule, instead recognizing damaged sites in a distributive manner. The proposed studies will investigate the molecular mechanisms by which the proteins in this complex interact with each other and with damaged nucleosomal DNA to initiate the repair of pyrimidine dimers in normal human cells. XPA cells, and their defective endonuclease complex, will be used as a model system in which these interactions are deficient. The proteins within the normal DNA endonuclease complex will be identified and isolated. cDNAs coding for each of these proteins will be cloned, sequenced and expressed in cultured human cells to confirm their role in DNA repair processes. The chromosomal localization of the genes for each of these proteins will be determined by in situ hybridization. The presence of mutations in the cDNAs coding for these proteins in XPA cells will be analyzed. Both the normal and XPA proteins will be purified by overexpressing the cDNAs of each in E. coli. Specificity of binding of each of these proteins, separately and then in combination, to site directed pyrimidine dimers on nucleosomal and non-nucleosomal DNA will be compared. The influence of the normal and XPA chromatin-interacting protein on the mode of interaction of each endonuclease with damaged naked and nucleosomal DNA will be examined. This unique combination of approaches will allow us to obtain valuable insight into the mechanisms responsible for repair of UV radiation damage in DNA.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Radiation Study Section (RAD)
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University of Medicine & Dentistry of NJ
Schools of Medicine
United States
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