Spore photoproduct lyase from Bacillus subtilis spores is a novel iron-sulfur DNA repair enzyme which shares features with proteins such as class III anaerobic ribonucleotide reductases and pyruvate-formate lyases

Abstract

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged 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 natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia coli overexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by 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 anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition of S-adenosylmethionine as a cofactor.

FIG. 1

Spore UV resistance of B. subtilis strains carrying various engineered alleles of SP lyase expressed from the amyE locus. Strains used were WN385 (vector only; open circles), WN386 (wild-type SP lyase; open triangles), WN390 (C-terminal histidine-tagged SP lyase; closed circles), and WN417 (N-terminal histidine-tagged SP lyase; closed triangles).

FIG. 2

Kinetics of SP repair during germination of spores of B. subtilis WN356 (open circles) and WN417 (closed circles) in DSM containing 10 mM l-alanine. Before germination, spores were irradiated with UV to obtain the following amounts of SP (expressed as percentage of total thymine): WN356, 1.1% and WN417, 2.1%. Results shown are the averages ± standard deviations of two (WN356) or three (WN417) independent experiments.

FIG. 3

SDS–10% PAGE-Western blot analysis of SP lyase purification. The 43- and 30-kDa protein molecular mass markers were visualized by staining with Ponceau S. Lane 1, lysed spores of strain WN356; lane 2, lysed spores of strain WN417; lane 3, cell-free extract of strain WN417 after clarification by high-speed centrifugation; lane 4, flow-through from nickel-NTA column; lane 5, eluate from nickel-NTA column with 250 mM imidazole.

FIG. 4

UV-visible absorption spectrum of 10×His-tagged SP lyase (0.8 mg/ml) purified from the E. coli overexpression system before (thick line) and after (thin line) treatment with 1 mM dithionite (0°C, 30 min).

FIG. 5

In vitro assay of SP lyase activity from cell-free extracts of strain WN417 spores. Assays were performed as described in Materials and Methods, and results are reported as averages ± standard errors of the means (n = 4). Differences in SP level in reaction mixtures to which cofactors were added compared to the control reaction mixture with extract added alone were significant at P = 0.05 (∗), P = 0.01 (∗∗), or P = 0.001 (∗∗∗) as determined by analysis of variance.

FIG. 6

In vitro repair of SP by 6×His-tagged SP lyase purified from B. subtilis spores as a function of enzyme added in the absence (open circles) or presence (closed circles) of 2 mM AdoMet. Data are reported as averages ± standard errors of the means (n = 2).