Purification and characterization of human ribonuclease HII

Abstract

A ribonuclease H activity from human placenta has been separated by Ion exchange chromatography from the major RNase HI enzyme. Additional chromato-graphlc 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-ethylmalelmide. 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. Renaturatlon gel assays and gel filtration experiments proved a mono-meric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases ollgoribo-nucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-medlated effect of antisense oligodeoxynucleotides.