Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. Direct identification of Lys-212 as the active nucleophilic residue

The human Endonuclease III (hNTH1), a homolog of the Escherichia coli Enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length Enzyme (304 amino acid residues) was expressed as a Glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The Enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 x 10(4) and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the Km was 47 nM, and kcat was approximately 0.6/min, independent of whether DHU paired with G or A. The Enzyme carries out beta-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the Enzyme. However, replacement with Arg-212 yielded an active Enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5'-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.