Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycos ylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer , displaying no detectable activity on oligonucleotides with trans-syn I, t rans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyase s that sequentially cleave the N-glycosyl bond of the 5' pyrimidine of a cy clobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme ap pears to utilize a primary amine as the attacking nucleophile. The formatio n of a covalent enzyme-DNA imino intermediate is evidenced by the ability t o trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP si te-containing oligonucleotide, yielding beta- and delta-elimination product s. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for ris-syn pyrimidine dimers. (C) 2000 Elsevier Science B.V. All rights reserved.