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We report here a human immunodeficiency virus type 1 (HIV-1) recombinant
ribonuclease H (RNase H) domain engineered to contain an N-terminal tag for its
isolation by affinity chromatography. The purified protein is active in
hydrolyzing RNA-DNA hybrids in two separate in vitro assay systems. In light of
recent reports of similar HIV-1 RNase H domains which were enzymatically
inactive (Becerra, S. P., Clore, G. M., Gronenborn, A. M., Karlstrom, A. R.,
Stahl, S. J., Wilson, S.M., and Wingfield, P.T. (1990) FEBS Lett. 270, 76-80;
Hostomsky, Z., Hostomska, Z., Hudson, G. O., Moomaw, E. W., and Nodes, B. R.
(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1148-1152), our results suggest that
a stretch of 20-30 residues immediately upstream of the polymerase-RNase H
junction (residues 440-441 of HIV-1 reverse transcriptase) may be required for
productive binding and alignment of the hybrid RNA-DNA substrate. The active
HIV-1 RNase H domain is suitable for structural analysis, thereby providing a
unique active molecule to better understand the structural basis for the
functional organization of RNase associated with the HIV-1 reverse transcriptase.
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