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J. Biol. Chem., Vol. 281, Issue 36, 25994-26003, September 8, 2006

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Characterization of an Active Spore Photoproduct Lyase, a DNA Repair Enzyme in the Radical S-Adenosylmethionine Superfamily^*

Jeffrey M. Buis, Jennifer Cheek¹, Efthalia Kalliri, and Joan B. Broderick²

From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717 and the Department of Chemistry, Michigan State University, East Lansing, Michigan 48824

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains 3.1 iron and 3.0 acid-labile S^2– per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM^–1 cm^–1) and 450 nm (10.5 mM^–1 cm^–1)). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]¹⁺ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]¹⁺ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 µmol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-³H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated -scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.

Received for publication, April 25, 2006 , and in revised form, June 27, 2006.

^* This work was supported in part by National Institutes of Health Grant GM67804 (to J. B. B.) and by start-up funds provided by Montana State University (to J. B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of Individual NRSA Grant GM20315 from the National Institutes of Health.

² To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Montana State University, Gaines Hall 108, Bozeman, MT 59717. Tel.: 406-586-2041; Fax: 406-994-5407; E-mail: jbroderick{at}chemistry.montana.edu.

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