Endoribonuclease activity of human apurinic/apyrimidinic endonuclease 1 revealed by a real-time fluorometric assay.
Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme with a well-established abasic DNA cleaving activity in the base excision DNA repair pathway and in providing redox activity to several well-known transcription factors. APE1 has recently been shown to cleave at the UA, CA, and UG sites of c-myc RNA in vitro and regulates c-myc messenger RNA (mRNA) in cells. To further understand this new endoribonuclease activity of APE1, we have developed an accurate, sensitive, and rapid real-time endonuclease assay based on a fluorogenic oligodeoxynucleotide substrate with a single ribonucleotide. Using this substrate, we carried out the first kinetic analysis of APE1 endoribonuclease activity. We found that the purified native APE1 cleaves the fluorogenic substrate efficiently, as revealed by a k(cat)/K(m) of 2.62x10(6)M(-1)s(-1), a value that is only 71-fold lower than that obtained with the potent bovine pancreatic RNase A. Ion concentrations ranging from 0.2 to 2mM Mg2+ promoted catalysis, whereas 10 to 20mM Mg2+ was inhibitory to the RNA-cleaving activity of APE1. The monovalent cation K+ was inhibitory except at 20mM, where it significantly stimulated recombinant APE1 activity. These results demonstrate rapid and specific endoribonucleolytic cleavage by APE1 and support the notion that this activity is a previously undefined function of APE1.
Kim
Sang-Eun
SE
Chemistry Program, University of Northern British Columbia, Prince George, BC, Canada V2N 4Z9.
Gorrell
Andrea
A
Rader
Stephen D
SD
Lee
Chow H
CH
D000818:Animals; D002417:Cattle; D053837:DNA Cleavage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase