Molecular characterization of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase
AdvisorNicholson, Wayne L.
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PublisherThe University of Arizona.
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AbstractSpore photoproduct lyase (SP lyase), is one of two DNA repair enzyme systems active during germination of Bacillus subtilis endospores. The presence of this enzyme is partially responsible for the high resistance of endospores to killing by ultraviolet (UV) radiation. Amino acid sequence analysis revealed that SplB contains three closely-spaced cysteine residues located in the amino-terminal one-third of the deduced sequence, which led to the hypothesis that SP lyase contains an iron-sulfur center. Chemical analysis and UV-visible spectra of purified SplB overproduced in E. coli supported this hypothesis. SP lyase activity requires an anaerobic/reducing environment and S-adenosylmethionine (AdoMet) as a cofactor. Identification of the catalytic requirements led to the formulation of the hypothesis that SP lyase effects catalysis via an adenosyl radical, which is generated by reduction of AdoMet by the iron-sulfur center. The electron paramagnetic resonance (EPR) spectrum of SplB purified under aerobic conditions was characteristic of a protein containing a partially degraded [3Fe-4S] center. When SplB was incubated with dithionite, the spectrum shifted to that of a reduced [4Fe-4S] center. When the enzyme was treated with AdoMet, there was a significant decrease in EPR signal intensity. These results indicate that the [4Fe-4S] center is capable of acquiring electrons and transferring them to AdoMet. Analysis of the reactions via high-pressure liquid chromatography and electrospray ionization mass spectroscopy showed that SP lyase generates 5'-deoxyadenosine during the reaction. Analysis of SplB via SDS-PAGE after treatment with the protein cross-linking agent dimethyl suberimidate and size exclusion chromatography under native conditions, revealed the presence of a protein species of approximately two times the molecular weight of SplB demonstrating that SplB is capable of forming dimers. Based on the evidence presented here, the following model has been generated: (i) two SplB units form a dimer that is coordinated via a [4Fe-4S] center; (ii) the [4Fe-4S] center becomes reduced and in turn reduces a molecule of AdoMet; (iii) AdoMet is split to generate an adenosyl radical which can then participate in the in situ reversal of SP into two intact thymines.
Degree ProgramGraduate College
Veterinary Science and Microbiology