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,Thomas Carell

doi:10.1074/jbc.m607053200

Abstract

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.

Highlights

Noteworthy is the unusually high stability of spores in the presence of UV light
Using a novel assay with a synthetic dinucleotide spore DNA lesion (SP) lesion substrate and with small DNA single strands, which contain one SP lesion at a defined site, we show that the fully reconstituted protein is active in the presence of AdoMet
The repair mechanism has been studied with the enzyme isolated from B. subtilis

Summary

Introduction

Noteworthy is the unusually high stability of spores in the presence of UV light. Using a novel assay with a synthetic dinucleotide SP lesion substrate and with small DNA single strands, which contain one SP lesion at a defined site, we show that the fully reconstituted protein is active in the presence of AdoMet. We observed a specific activity of 2.6 ␮mol of SP repaired per min/mg of SplG. Assay for ssDNA Repair, Cleavage of AdoMet, Inhibition with aza-AdoMet and the 5S-and 5R-configured Spore Lesion—To prove enzyme activity, the amount of 5Ј-deoxyadenosine and repaired SP was determined and analyzed by rp-HPLC.

Results

Conclusion