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

The predominant photolesion in the DNA of UV-irradiated dormant bacterial s pores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP), A major determinant of SP repair during spo re germination is its direct reversal by the enzyme SP base, encoded by the splB gene in Bacillus subtilis. SpIB protein containing an N-terminal tag of six histidine residues [(6His)SpIB] was purified from dormant B, subtili s spores and shown to efficiently cleave SP but not cyclobutane cis,syn thy mine-thymine dimers in vitro. In contrast, SpIB protein containing an N-ter minal 10-histidine tag [(10His)SpIB] purified fi om an Escherichia coil ove rexpression system was incompetent to cleave SP unless the 10-His tag was f irst removed by proteolysis at an engineered factor Xa site. To assay the p arameters of binding of SpIB protein to UV-damaged DNA, a 35-bp double-stra nded oligonucleotide was constructed which carried a single pair of adjacen t thymines on one strand. Irradiation of the oligonucleotide in aqueous sol ution or at 10% relative humidity resulted in formation of cyclobutane pyri midine dimers (Py lozenge Py) or Sp, respectively. (10His)SpIB was assayed for oligonucleotide binding using a DNase I protection assay. In the presen ce of (10His)SpIB, the SP-containing oligonucleotide was selectively protec ted from DNase I digestion (half-life, >60 min), while the Py lozenge Py-co ntaining oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footpr inting of (10His)SpIB bound to the artificial substrate was carried out uti lizing the P-32 end-labeled 35-bp oligonucleotide containing SP, DNase I fo otprinting showed that SpIB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensiti ve sites within the protected region. (10His)SpIB also caused significant e nhancement of DNase I digestion of the SP-containing oligonucleotide for at least a full helical turn 3' to the protected region. The data suggest tha t binding of SP base to SP causes significant bending or distortion of the DNA helix in the vicinity of the lesion.