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Nucleic Acids Research, 1994, Vol. 22, No. 24 5247-5254
© 1994

Articles

Purification and characterization of human ribonuclease HII

Peter Frank+, Sylvie Albert§, Christian Cazenave and Jean-Jacques Toulme*

Laboratoire de Biophysique Moleculaire INSERM U 386, Universite de Bordeaux II 146 Rue Leéo Saignat, F-33076 Bordeaux cedex, France

*To whom correspondence should be addressed

+Present addresses: 1nsütute for Tumor BioIo', Borschkegasse 8a, A-1090 Wien, Austria

§Universitnumero de Perpignan, Laborazoire de Phystologie et Biologie Moléculaire Végétales, 52 Avenue de Villeneuve, 66860 Perpignan cédex, France

Received September 16, 1994. Accepted November 7, 1994.

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.


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