Mechanism of action of a mammalian DNA repair endonuclease.
The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.
Doetsch
P W
PW
Helland
D E
DE
Haseltine
W A
WA
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004262:DNA Restriction Enzymes / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D009699:N-Glycosyl Hydrolases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase