Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5-thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV-irradiated DNA

Abstract

The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py lozengePy) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py lozengePy-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the (32)P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest that binding of SP lyase to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.

FIG. 1

Assay of SP cleavage activity on SP-containing B. subtilis chromosomal DNA from UV-irradiated spores. The proteins assayed were (6His)SplB purified from spores of B. subtilis strain WN417 (triangles), (10His)SplB overproduced in and isolated from E. coli (open circles), and (10His)SplB purified from E. coli and cleaved with factor Xa (solid circles). The data are averages ± standard deviation of duplicate determinations.

FIG. 2

Kinetics of reversal of SP (solid circles) or cis,syn T◊T (open circles) by (6His)SplB protein (2 μg) purified from spores of B. subtilis strain WN417. The initial amounts of photoproducts in each reaction mixture were 2.68% SP and 2.72% T◊T (expressed as a percentage of total thymine).

FIG. 3

DNase I protection experiment. The 35-bp double-stranded oligonucleotide carrying: no dimer (A), Py◊Py (B), or SP (C) was preincubated either with (solid symbols) or without (open symbols) (10His)SplB and then treated with DNase I and quantitated as described in Materials and Methods. The data are represented as averages ± standard deviations of three independent experiments. The horizontal dashed line represents 50% degradation.

FIG. 4

DNase I footprinting experiment. (A) Autoradiogram. Lane 1, untreated SP-containing 35-nt oligonucleotide; lanes 2 to 5, SP-containing oligonucleotide after partial DNase I digestion in the absence (lane 2) or the presence of 5.0 (lane 3), 2.5 (lane 4), or 0.5 μg (lane 5) of (10His)SplB. The molar ratios of (10His)SplB to oligonucleotide were 10:1 (lane 3), 5:1 (lane 4) and 1:1 (lane 5). The two thymines corresponding to the position of SP are shown in boldface and indicated on the autoradiogram by arrowheads. (B) Densitometric scan of lane 2 (thin line) and lane 3 (thick line) of the autoradiogram displayed in panel A. (C) Quantitative summary of DNase I footprinting experiments. The protection (upward bars) or enhancement (downward bars) of DNase I digestion at each base on the SP-containing oligonucleotide by binding of 2.5 (open bars) and 5.0 μg (solid bars) of (10His)SplB was determined relative to the unbound oligonucleotide. The positions of the two DNase I-hypersensitive sites within the footprint are denoted by asterisks. The position of SP is denoted by the two open T residues at coordinates 19 and 20. The data using 5.0 μg of (10His)SplB are the averages of two independent determinations; the error bars denote the maximum value obtained.

FIG. 5

Proposed sequence of events in SP cleavage by SP lyase. Abbreviations: Ado, adenosine; FeS, iron-sulfur cluster; met, methionine. See the text for details.