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Poster

Spore Photoproduct Lyase and its Substrate in double stranded Oligonucleotides

Andrea C. Kneuttinger, Korbinian Heil, Dr. Sabine Schneider, Prof. Dr. Thomas Carell1
1 Centre for Integrative Protein Science at the Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Butenandtstr. 5-13, 81377 Munich, Germany; e-mail: thomas.carell@cup.uni-muenchen.de

Abstract

Radical SAM enzymes are ubiquitous enzymes involved in a rich variety of functions.1 One of these [4Fe-4S] cluster containing proteins is the spore photoproduct lyase (SPL). This enzyme catalyzes the repair of the spore photoproduct (SP), a UV-induced DNA lesion that occurs in bacterial spores. Theoretically, four different isomers of this dithymidine (TpT) derived lesion can be formed. However, only one is found in nature and repaired by the SPL. This is the 5R SP lesion formed in 5´→ 3´ direction.2-3 Up to date, only few publications have shed light on the repair mechanism. The main unanswered questions concern the details of the radical catalysis and the role of S-adenosylmethionine (SAM).

To explore the interaction between this enzyme and its substrate we use a His6-tagged SPL derived from Geobacillus stearothermophilus and chemically synthesized SP analogs that were incorporated into oligonucleotides.4 The anaerobically purified SPL holds an intact [4Fe-4S] cluster as shown by UV-VIS spectra. In in vitro experiments we could confirm the exclusive repair of the 5R isomer of the SP lesion. Additionally, co-crystallization experiments with a polymerase allowed us to further characterize the base pairing and duplex distorsion properties of the SP lesion.5 Biochemical assays to address the role of SAM in the catalytic repair reaction and to characterize SPL enzyme activity are undergoing.

Formation and repair of SP.

References

  1. P. A. Frey, O. Th. Magnusson, Chem. Rev. 2003, 103, 2129-2148
  2. C. Mantel, A. Chandor, D. Gasparutto, T. Douki, M. Atta, M. Fontecave, P.-A. Bayle, J.-M. Mouesca, M. Bardet, J. Am. Chem. Soc. 2008, 130, 16978-16984
  3. T. Chandra, S. C. Silver, E. Zilinskas, E. M. Shepard, W. E. Broderick, J. B. Broderick, J. Am. Chem. Soc. 2009, 131, 2420-2421
  4. E. Bürckstümmer, T. Carell, Chem. Commun. 2008, 4037-4039
  5. K. Heil, A. C. Kneuttinger, S. Schneider, U. Lischke, T. Carell, Chem. Eur. J., 2011, in print

DOI®: 10.3288/contoo.paper.1422
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