AN ACTIVE RECOMBINANT P15 RNASE-H DOMAIN IS FUNCTIONALLY DISTINCT FROM THE RNASE-H DOMAIN ASSOCIATED WITH HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE

An active p15 RNase H domain, consisting of amino acids 427-560 of hum an immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) an d a genetically engineered penta-histidine N-terminal affinity tag, wa s expressed in Escherichia coli and purified to apparent homogeneity b y immobilized metal affinity chromatography. The purified p15 RNase H domain exhibited no substrate preference far [H-3]poly(rG).poly(dC) co mpared to [H-3]poly(rA).poly(dT), in contrast with the HIV-1 RT-associ ated RNase H, which showed a 30-fold preference for the former substra te. Unlike the HIV-1 RT-associated RNase H, when challenged with unlab eled substrate, the recombinant p15 RNase H domain was relatively nonp rocessive in RNA degradative activity of the [H-3]poly(rA).poly(dT) du plex. kinetic studies using p15 RNase H showed substrate inhibition wi th an apparent K-i value of 0.12 mu M for the [H-3]poly(rA).poly(dT) h ybrid. Substrate inhibition was not observed for the HIV-1 RT-associat ed RNase H. The results show that the isolated p15 HIV-1 RNase H domai n is functionally distinct from the recombinant HIV-1 RT-associated RN ase H.