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Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain.
Smith JS; Roth MJ; Department of Biochemistry, Robert Wood Johnson
September 30, 1993
J Virol. 1993 Jul;67(7):4037-49. Unique Identifier : AIDSLINE

We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.

Amino Acid Sequence Base Sequence Cloning, Molecular HIV-1/*ENZYMOLOGY Molecular Sequence Data Oligodeoxyribonucleotides/CHEMISTRY Ribonuclease H, Calf Thymus/CHEMISTRY/*ISOLATION & PURIF RNA-Directed DNA Polymerase/*CHEMISTRY RNA, Transfer, Lys/METABOLISM RNA, Viral/METABOLISM Structure-Activity Relationship Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. JOURNAL ARTICLE