PURIFICATION AND CLONING OF MICROCOCCUS-LUTEUS ULTRAVIOLET ENDONUCLEASE, AN N-GLYCOSYLASE ABASIC LYASE THAT PROCEEDS VIA AN IMINO ENZYME-DNA INTERMEDIATE

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V fro m bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol , Chem, 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol, Chem, 257, 13465-13474), this study describes three i ndependent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa cont aminant copurified with the doublet through many of the chromatographi c steps, but it was determined to be a homolog of Escherichia coli rib osomal protein L6. Edman degradation analyses of the active proteins y ielded identical NH2-terminal amino acid sequences, The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned, The protein bears strong sequence similarities to the E. coli repair proteins endonucle ase III and MutY. Nonetheless, traditionally purified M. luteus protei n acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, ( 6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch, The UV endonuclease incised cis-syn dimer-containing DN A in a dose-dependent manner and exhibited linear kinet ics within tha t dose range, Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme su bstrate product, These last findings confirm that the catalytic mechan ism of M. luteus UV endonuclease, like those of other glycosylase/AP l yases, involves an imino intermediate.