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 pyri midines in DNA. Its cDNA was cloned, and the full-length enzyme (304 a mino acid residues) was expressed as a glutathione S-transferase fusio n polypeptide in E. coli. Purified wild-type protein with two addition al amino acid residues and a truncated protein with deletion of 22 res idues at the NH, terminus were equally active and had absorbance maxim a at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]clust er, as in E. coli Nth. The enzyme cleaved thymine glycol-containing fo rm 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 subs trate, the K-m was 47 nM, and k(cat) was similar to 0.6/min, independe nt of whether DHU paired with G or A. The enzyme carries out p-elimina tion and forms a Schiff base between the active site residue and the d eoxyribose generated after base removal. The prediction of Lys-212 bei ng the active site was confirmed by sequence analysis of the peptide-o ligonucleotide adduct, Furthermore, replacing Lys-212 with Gin inactiv ated the enzyme. However, replacement with Arg-212 yielded an active e nzyme with about 85-fold lower catalytic specificity than the wild-typ e protein. DNase I footprinting with hNTH1 showed protection of 10 nuc leotides centered around the base lesion in the damaged strand and a s tretch of 15 nucleotides (with the G opposite the lesion at the 5'-bou ndary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, locali zed in both the nucleus and the cytoplasm.