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

The major DNA photoproduct of dormant, UV-irradiated Bacillus subtilis spares is the thymine dieter 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)). During spore germination, SP is reversed to two intact thymines in situ by the DNA repair enzyme SP lyase, an S-adenosyimethionine (S-AdoMet)-dependent iron-sulfur ([Fe-S]) protein encoded by the splB gene. In the present work, cross-linking, SDS/PAGE, and size exclusion chromatography revealed that SpIB protein dimerized when incubated with iron and sulfide under anaerobic reducing conditions. SpIB isolated under aerobic conditions generated 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 [4Fe-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 donating electrons to S-AdoMet to generate a 5 ' -adenosyl radical that is then used for the in situ reversal of SP. Thus, SP lyase belongs to the "radical SAM" superfamily of enzymes that use [Fe-S] centers and S-AdoMet to generate adenosyl radicals to effect catalysis. SP lyase is unique in being the first and only DNA repair enzyme known to function via this novel enzymatic mechanism.