Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide.
We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.
Hilbert
T P
TP
Department of Pathology, New York University Medical Center, New York City, New York 10016, USA.
Boorstein
R J
RJ
Kung
H C
HC
Bolton
P H
PH
Xing
D
D
Cunningham
R P
RP
Teebor
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003432:Cross-Linking Reagents; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D066298:In Vitro Techniques; D008190:Lyases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C029389:thymine glycol; 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; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D013941:Thymine