Purification and characterization of human ribonuclease HII.
A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.
Frank
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Laboratoire de Biophysique Moléculaire, INSERM U 386, Université de Bordeaux II, France.
Albert
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Cazenave
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C
Toulmé
J J
JJ
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009696:Nucleic Acids / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D009696:Nucleic Acids; C091139:ribonuclease HII; D016914:Ribonuclease H; D008274:Magnesium; D005033:Ethylmaleimide