This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ultraviolet (UV) light is known to cause a number of effects after absorption by living cells, such as genetic mutation, cell death and cellular transformation. These latter types of damage are mediated, at least in part, by photoproducts formed in the genomic DNA of the cell. One main focus of our work is on the role of the minor base 5-methylcytosine (m5C) in mediating damage induced by absorption of light, although investigations of the reactivity of thymine (Thy), cytosine (Cyt) and halogenated uracils (e.g. 5-bromouracil) are also being carried out. Our primary aim is directed towards isolating and characterizing a number of photoproducts that we believe could be relevant to achieving a chemical understanding of UV-induced DNA damage. Among the compounds of current interest are the (6-4) adducts and Dewar adducts of m5C with Cyt and Thy, both in nucleobase and nucleoside form and on adducts formed with molecules in the environment of DNA in the cell (e.g. water, amino acids in proteins). These compounds are being prepared utilizing procedures that avoid conditions under which these compounds are unstable. High performance liquid chromatography (HPLC) is being used to isolate the various compounds in a pure state. They are being structurally characterized using proton and carbon-13 NMR spectroscopy, UV spectroscopy, electrospray ionization and MALDI mass spectrometry and, where appropriate, circular dichroic spectroscopy. Their chemical stability under conditions of DNA digestion is also being determined.
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