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J. Biol. Chem., Vol. 281, Issue 47, 36317-36326, November 24, 2006

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Characterization of a New Thermophilic Spore Photoproduct Lyase from Geobacillus stearothermophilus (SplG) with Defined Lesion Containing DNA Substrates^*

J. Carsten Pieck, Ulrich Hennecke, Antonio J. Pierik, Marcus G. Friedel, and Thomas Carell¹

From the Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich and the Department of Microbiology, Philipps University Marburg, D-35032 Marburg, Germany

The Geobacillus stearothermophilus splG gene encodes a thermophilic spore photoproduct lyase (SplG) that belongs to the family of radical S-adenosylmethionine (AdoMet) enzymes. The aerobically purified apo-SplG forms a homodimer, which contains one [4Fe-4S] cluster per monomer unit after reconstitution to the holoform. Formation of the [4Fe-4S] cluster was proven by quantification of the amount of iron and sulfur per homodimer and by UV and EPR spectroscopy. The UV spectrum features a characteristic absorbance at 420 nm typical for [4Fe-4S] clusters, and the EPR data were found to be identical to those of other proteins containing an [4Fe-4S]⁺ center. Probing of the activity of the holo-SplG with oligonucleotides containing one spore photoproduct lesion at a defined site proved that the enzyme is able to turn over substrate. In addition to repair, we observed cleavage of AdoMet to generate 5'-deoxyadenosine. In the presence of aza-AdoMet the SplG is completely inhibited, which provides direct support for the repair mechanism.

Received for publication, July 25, 2006 , and in revised form, September 11, 2006.

^* This work was supported by Deutsche Forschungsgemeinschaft, Volkswagen Foundation, and the European Union Marie Curie Network (CLUSTOX DNA). 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 To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany. Tel.: 49-89-2180-77750; Fax: 49-89-2180-77756; E-mail: thomas.carell{at}cup.uni-muenchen.de.

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