SPORE PHOTOPRODUCT LYASE FROM BACILLUS-SUBTILIS SPORES IS A NOVEL IRON-SULFUR DNA-REPAIR ENZYME WHICH SHARES FEATURES WITH PROTEINS SUCH ASCLASS-III ANAEROBIC RIBONUCLEOTIDE REDUCTASES AND PYRUVATE-FORMATE LYASES

The major photoproduct in W-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore ph otoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP durin g spore germination by the DNA repair enzyme SP lyase. To study the mo lecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of s ix histidine residues at its amino terminus. The engineered six-His-ta gged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion -insertion mutation which removed the entire splAB operon at its natur al locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spo res and from an Escherichia coli overexpression system by nickel-nitri lotriacetic acid (NTA) agarose affinity chromatography and was shown b y Western blotting, UV visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaer obic ribonucleotide reductases and pyruvate-formate lyases. SP lyase w as capable of reversing SP from purified SP-containing DNA in an in vi tro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase a ctivity was dependent upon reducing conditions and addition of S-adeno sylmethionine as a cofactor.