Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin.
An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.
Kim
Y S
YS
Department of Biochemistry, Yonsei University College of Medicine, Seoul, Korea.
Kim
J W
JW
Lee
S E
SE
Oh
S H
SH
D000818:Animals; D001665:Binding Sites; D002843:Chromatin / Q000201:enzymology*; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D002843:Chromatin; D013438:Sulfhydryl Compounds; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008274:Magnesium