MECHANISTIC STUDIES OF THE FE/S ENZYME SP LYASE

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The specific aim of this proposal is to develop a mechanistic understanding of how spore photoproduct (SP) lyase, an adenosylmethione-dependent iron-sulfur enzyme, repairs DNA. SP lyase will first by cloned, over-expressed in E. coli, and then purified. The iron- sulfur cluster of SP lyase will be characterize with spectroscopic techniques including electronic absorption, magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, Mossbaur spectroscopy. Am activity assay will be developed to probe the necessity for the iron-sulfur cluster, S-adenosylmethionine, and reducing agents on the enzyme activity. The binding of SP lyase to DNA will be determined using two different methods, DNA footprinting and the gel shift assay. The binding of SP lyase to DNA will be determined using two different methods, DNA footprinting and the gel shift assay. The effect on DNA binding of the presence or absence of various co-factors will also be examined by these methods. Rapid freeze-quench techniques will be used to spectroscopically probe reaction intermediates, including the possibility of radical intermediates. Ultimately, the goal is to gain an understanding of the mechanism by which SP lyase repairs thymine dimers, including the detailed roles of the iron-sulfur cluster and adenosylmethione.