SUBSTITUTION OF A HIGHLY BASIC HELIX LOOP SEQUENCE INTO THE RNASE-H DOMAIN OF HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE RESTORES ITS MN2-DEPENDENT RNASE-H ACTIVITY()

Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a mul tifunctional protein, containing both DNA polymerase and RNase H activ ity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RN A strand of RNA-DNA hybrids. While the domain that carries out the RNa se H activity in HIV RT can be expressed as an independent, folded pol y-peptide, it is inactive as an RNase H. Here, we report the overexpre ssion and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 bas ic helis/loop has been substituted for the corresponding sequence of H IV RNase H. The resulting polypeptide (RNH102) has Mn2+-dependent RNas e H activity and is more stable than the independently expressed wild- type HIV RNase H domain.