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.
Ikeda
S
S
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Biswas
T
T
Roy
R
R
Izumi
T
T
Boldogh
I
I
Kurosky
A
A
Sarker
A H
AH
Seki
S
S
Mitra
S
S
D001483:Base Sequence; D001894:Borohydrides; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002384:Catalysis; D018983:DNA Footprinting; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D001894:Borohydrides; D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins; C009282:sodium cyanoborohydride; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008239:Lysine