PURIFICATION AND CHARACTERIZATION OF HUMAN NTH1, A HOMOLOG OF ESCHERICHIA-COLI ENDONUCLEASE-III - DIRECT IDENTIFICATION OF LYS-212 AS THE ACTIVE NUCLEOPHILIC RESIDUE
S. Ikeda et al., PURIFICATION AND CHARACTERIZATION OF HUMAN NTH1, A HOMOLOG OF ESCHERICHIA-COLI ENDONUCLEASE-III - DIRECT IDENTIFICATION OF LYS-212 AS THE ACTIVE NUCLEOPHILIC RESIDUE, The Journal of biological chemistry, 273(34), 1998, pp. 21585-21593
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.