Characterization of a new thermophilic spore photoproduct lyase from Geobacillus stearothermophilus (SplG) with defined lesion containing DNA substrates.
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.
Pieck
J Carsten
JC
Department of Chemistry and Biochemistry, Ludwig Maximilians University Munich, D-81377 Munich, Germany.
Hennecke
Ulrich
U
Pierik
Antonio J
AJ
Friedel
Marcus G
MG
Carell
Thomas
T
D001406:Bacillaceae / Q000502:physiology*; D002384:Catalysis; D002853:Chromatography, Liquid; D004247:DNA / Q000737:chemistry*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D007506:Iron-Sulfur Proteins; D009841:Oligonucleotides; D011506:Proteins; D004247:DNA; C088014:spore photoproduct lyase