DIVALENT-CATION MODULATION OF THE RIBONUCLEASE FUNCTIONS OF HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE-TRANSCRIPTASE

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H ) functions of HIV-1 reverse transcriptase (RT) has been evaluated usi ng a model 90-nt RNA template/36-nt DNA primer, Wild type enzyme exhib its similar endonuclease and directional processing activities in resp onse to both cations, while RNase H activity (hydrolysis of double-st randed RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu(478) --> Gln(478)), which participates in metal ion binding, is completely inactive in Mg2+, However, Mn2+ rest ores specifically its endoribonuclease activity. the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication pri mer, eliminating the possibility of contaminating Escherichia coli RNa se H in our recombinant enzyme. However, the efficiency with which mut ant RT catalyzes transfer of nascent DNA between RNA templates (an eve nt mandating RNase H activity) is severely reduced. These findings rai se the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retrovira l replication.