The ultraviolet component of sunlight is one of the constant threats to the integrity of the genetic material and the survival of species. With the gradual depletion of the stratospheric ozone the fraction of UV-B (290-320 nm) reaching the biosphere is increasing, with serious consequences for humans and other organisms. The most important cellular target of UV is DNA. The two major lesions produced in DNA by UV are the cyclobutane pyrimidine dimer (Pyr<>Pyr) and the (6-4) pyrimidine- pyrimidone photoproduct (6-4 photoproduct). The Pyr<>Pyr and (6-4) photoproducts kill cells by blocking replication and transcription. On rare occasions when the DNA is replicated past the lesion mutations arise which may cause cancer and other unfavorable phenotypes. Both the Pyr<>Pyr and the (64) photoproduct are eliminated from DNA by repair enzymes. DNA photolyase is a repair enzyme which utilizes the energy of near UV- visible light to convert Pyr<>Pyr to monomers and thus prevent the harmful effects of solar UV. The enzyme has a flavin and a folate cofactors. We wish to understand how the enzyme recognizes damage and how it converts light energy into chemical energy. Towards this goal the following experiments will be conducted. (1) The structure of the photolyase from Escherichia coli will be solved by X-ray crystallography. The positions and orientations of the two photolyase cofactors relative to one another will be determined by NMR spectroscopy. Enzyme-substrate crystals will be made and the structure of the complex will be solved. (2) Using site- directed mutagenesis, the roles of the amino acids in the active center will be investigated with regard to their contributions to DNA binding, and energy and electron transfer reactions which ultimately repair DNA. (3) The blue-light photoreceptor which is highly homologous to photolyase but has no photolyase activity will be purified and characterized and its mutant forms will be generated to gain insight into the evolutionary origin and structures of these two closely related proteins of vastly different functions. (4) The reaction intermediates of photolyase will be identified by using picosecond laser flash photolysis with substrates of different pyrimidine compositions. Reaction intermediates corresponding to both the substrate and the catalytic flavin cofactor will be identified in order to understand the mechanisms of forward and back-electron transfer. (5) Recent evidence indicates that the (6-4) photoproduct is repaired by another light-dependent enzyme. This, so-called (6-4) photolyase will be purified by standard biochemical methods and its reaction mechanism will be solved by using synthetic substrates and fast-reaction techniques.
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