CLONING AND EXPRESSION OF THE CDNA-ENCODING THE HUMAN HOMOLOG OF THE DNA-REPAIR ENZYME, ESCHERICHIA-COLI ENDONUCLEASE-III

We previously purified a bovine pyrimidine hydratethymine glycol DNA g lycosylase/AP lyase, The amino acid sequence of tryptic bovine peptide s was homologous to Escherichia coli endonuclease III, theoretical pro teins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and hu man 3'-expressed sequence tags (3'- ESTs) (Hilbert, T. P., Boorstein, R, J., Kung, H, C., Bolton, P. H., X ing, D., Cunningham, R. P., Teebor, G. W. (1996) Biochemistry 35, 2505 -2511). Now the human 3'-EST was used to isolate the cDNA clone encodi ng the human enzyme, which, when expressed as a GST-fusion protein, de monstrated thymine glycol-DNA glycosylase activity and, after incubati on with NaCNBH3, became irreversibly cross linked to a thymine glycol- containing oligodeoxynucleotide, a reaction characteristic of DNA glyc osylase/AP lyases. Amino acids within the active site, DNA binding do mains, and [4Fe-4S] cluster of endonuclease III are con served in the human enzyme. The gene for the human enzyme was localized to chromosom e 16p13.2-.3. Genomic sequences encoding putative endonuclease III hom ologues are present in bacteria, archeons, and eukaryotes. The ubiquit ous distribution of endonuclease III-like proteins suggests that the 5 ,6-double bond of pyrimidines is subject to oxidation, reduction, and/ or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism.