Kinetic, mechanistic and spectroscopic studies of spore photoproduct lyase
Silver, Sunshine Christine
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Spore forming organisms are a health threat to humans and other animals in part due to a remarkable resistance to UV irradiation. This resistance results from two events: first, the formation of a unique thymine dimer, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP) upon UV irradiation; and more importantly, the rapid and specific repair of this DNA photoproduct to two thymines by spore photoproduct lyase (SP lyase). Understanding the molecular basis of this radical-mediated DNA repair will ultimately allow for a better understanding of how to address the health risks caused by spore forming bacteria. SP lyase requires S-adenosyl-L-methionine and a [4Fe-4S] ¹+/²+ cluster to perform its catalysis. Presented in this work is a characterization of Clostridium acetobutylicm SP lyase and its ability to repair stereochemically defined dinucleoside and dinucleotide synthetic substrates. Careful synthesis and characterization followed by assays monitored by HPLC indicate SP lyase repairs only the 5R isomer of SP with an activity of 0.4 nmol/min/mg (dinucleoside substrate) and 7.1 nmol/min/mg (dinucleotide substrate). These results support the longstanding theory of SP formation by dimerization of adjacent thymines in double-helical DNA. Kinetic and mechanistic studies were pursued to further elucidate the mechanism of SP repair. Upon pre-reducing SP lyase, the specific activity increased nearly 4-fold to 1.29 umol/min/mg. Mechanistic studies utilizing [C-6- ³ H] SP DNA as the substrate revealed a primary tritium kinetic isotope effect of 16.1, indicating a rate determining step during the repair reaction. These results suggest nonstereospecific SP formation regarding the C-6 position and subsequent stereospecific abstraction of the C-6 H atom by SP lyase. Mössbauer and Fe/S K-edge X-ray absorption studies of anaerobically prepared SP lyase aided in further characterization of the [4Fe-4S] cluster and its interaction with SAM. The Fe K-edge EXAFS provide evidence for a slight cluster distortion upon interacting with SAM as a new spectral feature indicative of longer Fe-Fe distances is observed. The Fe K-edge XANES provide further support that SAM is not undergoing reductive cleavage in the presence of reduced SP lyase. Our XAS studies may provide new insights into the mechanism by which radical SAM enzymes initiate their diverse catalysis.