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

Abstract

The purification and characterization of a pyrimidine dimer-specific glycosylase/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, trans–syn II, (6–4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the Nglycosyl bond of the 5′ pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme–DNA imino intermediate is evidenced by the ability to 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 site-containing oligonucleotide, yielding β- and δ-elimination products. 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 cis–syn pyrimidine dimers.