Enzynnes utilize organic radicals to catalyze a variety of important nnetabolic reactions. The overall goal of this research is to delineate the mechanistic details of radical generation and control by these enzymes. This research in the ROO phase will focus on a DNA repair enzyme named spore photoproduct lyase (SPL). SPL utilizes S-adenosylmethionine (SAM) coupled by a unique [4Fe-4S] cluster to generate the reactive organic radicals to repair the unique T-T crosslink 5-thyminyl-5, 6-dihydrothymine (commonly called spore photoproduct, SP) formed upon UV irradiation. SPL exists in the spores of bacteria such as B. subtilis and B. anthracis. It adopts a """"""""direct reverse"""""""" strategy to repair SP, meaning that the UV damage is quickly reversed with neither removal nor replacement of the damaged thymine bases. It thus represents a unique DNA repair pathway in Nature. In addition, the efficient DNA repair catalyzed by SPL makes UV irradiation no longer lethal to the spore-forming bacteria. To understand the SPL mediated DNA repair reaction, chemical, kinetic, spectroscopic, and mutagenic methods will be employed. The objectives include: investigating the SPL activity using substrates with a wide range of DNA secondary structures, probing the reaction mechanism by SP analogues (mechanism-based enzyme inhibitors), and examining the kinetic isotope effects and reaction reversibility. In addition, the redox potential of the [4Fe-4S] cluster will be determined and the influence of SAIV! and key amino acids to the redox potential will be investigated. Understanding the enzyme mechanism will help us identify potential SPL inhibitors. As SPL is the key enzyme to repair the UV damage in endospore-forming bacteria, inhibiting its activity in vivo will prevent the bacteria from fixing these damages at the germination phase. In combination with the SPL inhibitor, UV irradiation will regain its power as a cheap and convenient tool for sterilization purpose.

Public Health Relevance

The efficient DNA repair ability represented by an enzyme named spore photoproduct lyase in endospore-forming bacteria is the key for their survival. Endospore-forming bacterial strains are responsible for a number of serious diseases in humans, including botulism (Clostridium botulinum), food poisoning (Glostridium perfringens), and anthrax (Bacillus anthracis). The mechanistic studies of snore nhotor^roduct Ivase should offer a nromisina wav to battle these deadly diseases.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Transition Award (R00)
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Reinlib, Leslie J
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Indiana University-Purdue University at Indianapolis
Schools of Arts and Sciences
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Yang, Linlin; Adhikari, Jagat; Gross, Michael L et al. (2017) Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyase. Photochem Photobiol 93:331-342
Yang, Linlin; Jian, Yajun; Setlow, Peter et al. (2017) Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme-The spore photoproduct lyase. DNA Repair (Amst) 53:31-42
Yang, Linlin; Li, Lei (2015) Spore photoproduct lyase: the known, the controversial, and the unknown. J Biol Chem 290:4003-9
Setlow, Peter; Li, Lei (2015) Photochemistry and Photobiology of the Spore Photoproduct: A 50-Year Journey. Photochem Photobiol 91:1263-90
Jian, Yajun; Ames, David M; Ouyang, Hao et al. (2015) Photochemical reactions of microcrystalline thymidine. Org Lett 17:824-7
Jian, Yajun; Lin, Gengjie; Chomicz, Lidia et al. (2015) Reactivity of damaged pyrimidines: formation of a Schiff base intermediate at the glycosidic bond of saturated dihydrouridine. J Am Chem Soc 137:3318-29
Ames, David M; Lin, Gengjie; Jian, Yajun et al. (2014) Unusually large deuterium discrimination during spore photoproduct formation. J Org Chem 79:4843-51
Lin, Gengjie; Jian, Yajun; Dria, Karl J et al. (2014) Reactivity of damaged pyrimidines: DNA cleavage via hemiaminal formation at the C4 positions of the saturated thymine of spore photoproduct and dihydrouridine. J Am Chem Soc 136:12938-46
Singh, Isha; Jian, Yajun; Lian, Yajun et al. (2014) The structure of an authentic spore photoproduct lesion in DNA suggests a basis for recognition. Acta Crystallogr D Biol Crystallogr 70:752-9
Jian, Yajun; Li, Lei (2013) Chemical syntheses of oligodeoxyribonucleotides containing spore photoproduct. J Org Chem 78:3021-9

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