This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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. Among these are 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. With the increasing importance of UVB light (between 290 and 320 nm) in the environment of living organisms, due to depletion of the ozone layer of the Earth, UV-induced lesions in DNA could play an increasing role in induction of skin cancer and other unwanted biological effects. The end result of the work described in this project would be increased understanding of what happens chemically to mammalian and plant DNA after it absorbs UVB light. In particular, the proposal focuses on the role of the minor base 5-methylcytosine (m5C) in mediating damage induced by absorption of UVB light. The present project has two component aims.
The first aim i s directed towards isolating and characterizing a number of photoproducts that we believe are relevant to achieving a chemical understanding of UVB-induced DNA damage. These compounds are the c,s and t,s mixed cyclobutane dimers , (6-4) adducts and Dewar adducts of m5C with thymine and cytosine, both in nucleobase and nucleoside form. To achieve this aim, the compounds will be prepared utilizing procedures that avoid conditions under which these compounds are unstable. High performance liquid chromatography (HPLC) will be used to isolate the various compounds in a pure state. They will then be 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 will also be determined.
The second Aim i s to determine if the various products isolated in experiments related to Aim 1 are also formed within the context of DNA and in the environment of a cell nucleus. Four types of system will be focussed on, namely calf thymus DNA and nuclei and wheat DNA and nuclei. The former DNA has a m5C content similar to human DNA (about 5% of DNA cytosines are methylated); the latter DNA has about 30% methylated cytosines. The protocols used for detection of these products in irradiated DNA include the following steps. After irradiation, protein will be removed and the DNA will be digested with gentle chemical nucleases. The digest will be examined by HPLC, to determine whether products are present that have identical retention times as those of authentic samples of the m5C products of interest; for this purpose, UV, fluorescence and mass spectral detection will be used to verify that the products of interest in the HPLC chromatogram have identical properties to those of authentic product. Yields of the various products will be determined and compared to published yields of corresponding photoproducts involving other pyrimidine base pairings. Experiments will also be conducted to determine if ureidoacrylonitrile and acrylamidine type compounds of m5C, such as isolated previously in my laboratory, are formed in these UVB irradiated systems.
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