The subunit structure and catalytic mechanism of the Bacillus subtilis DNArepair enzyme spore photoproduct lyase

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spar es is the thymine dieter 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)). During spore germination, SP is reversed to two intact thymines in s itu by the DNA repair enzyme SP lyase, an S-adenosyimethionine (S-AdoMet)-d ependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the pres ent work, cross-linking, SDS/PAGE, and size exclusion chromatography reveal ed that SpIB protein dimerized when incubated with iron and sulfide under a naerobic reducing conditions. SpIB isolated under aerobic conditions genera ted art EPR spectrum consistent with that of a partially degraded [3Fe-4S) center, and reduction of SpIB with dithionite shifted the spectrum to that of a [4Fe-4S) center. Addition of S-AdoMet to SpIB converted some of the [4 Fe-4Sj centers to an EPR-silent form consistent with electron donation to S -AdoMet. HPLC and electrospray ionization MS analyses showed that SP lyase cleaved S-AdoMet to generate 5 ' -deoxyadenosine. The results indicate that (i) SP lyase is a homodimer of SpIB; (ii) dieter formation is coordinated by a [4Fe-4S) center; and (iii) the reduced [4Fe-4S) center is capable of d onating electrons to S-AdoMet to generate a 5 ' -adenosyl radical that is t hen used for the in situ reversal of SP. Thus, SP lyase belongs to the "rad ical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to ge nerate adenosyl radicals to effect catalysis. SP lyase is unique in being t he first and only DNA repair enzyme known to function via this novel enzyma tic mechanism.