Purification and characterization of HeLa endonuclease R. A G-specific mammalian endonuclease.
We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.
Gottlieb
J
J
Department of Microbiology, State University of New York, Stony Brook Medical School 11794.
Muzyczka
N
N
D001482:Base Composition; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011068:Poly G / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D002413:Cations, Divalent; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D011068:Poly G; C026628:poly(dG); D008345:Manganese; D012965:Sodium Chloride; D011189:Potassium Chloride; C029603:deoxyguanosine triphosphate; D004247:DNA; D004706:Endodeoxyribonucleases; C064670:endonuclease R; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate; D002118:Calcium