Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis.
The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.
Chandor
Alexia
A
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DRDC-CB, UMR 5047, Commissariat à l'Energie Atomique/CNRS/Université Joseph Fourier, 17 Rue des Martyrs 38054, Grenoble Cedex 09, France.
Berteau
Olivier
O
Douki
Thierry
T
Gasparutto
Didier
D
Sanakis
Yannis
Y
Ollagnier-de-Choudens
Sandrine
S
Atta
Mohamed
M
Fontecave
Marc
M
D001412:Bacillus subtilis / Q000201:enzymology*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D011506:Proteins / Q000737:chemistry*; D011740:Pyrimidine Dimers / Q000737:chemistry; D013379:Substrate Specificity; D013941:Thymine / Q000737:chemistry; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D011506:Proteins; D011740:Pyrimidine Dimers; C088014:spore photoproduct lyase; D004255:Deoxyribodipyrimidine Photo-Lyase; D013941:Thymine