MECHANISM OF HIV-1 REVERSE-TRANSCRIPTASE - TERMINATION OF PROCESSIVE SYNTHESIS ON A NATURAL DNA-TEMPLATE IS INFLUENCED BY THE SEQUENCE OF THE TEMPLATE-PRIMER STEM

During processive DNA synthesis in vitro, the human immunoefficiency v irus, type 1 (HIV-1) reverse transcriptase encounters template nucleot ide positions at which continued synthesis is difficult. At these posi tions, the enzyme has a relatively high probability of dissociating fr om the template, and product molecules of corresponding length accumul ate as the incubation proceeds. These positions, which are known as te rmination sites, could be associated with template secondary structure s in some cases, but many termination sites appear to be template sequ ence-related rather than secondary structure-related. Mechanisms produ cing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. S everal general trends emerged from the study. First, strong terminatio n sites rarely correspond to dATP as the ''incoming'' substrate opposi te template T. Second, the sequence of the template-primer stem is mor e important for termination than the sequence of the single-stranded t emplate ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the templ ate-primer stem alters termination at other positions, a few nucleotid es distant at the primer 3' end. A and C as template bases in the temp late-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse t ranscriptase has properties reminiscent of a sequence-specific double- stranded DNA-binding protein in that its binding mechanism can disting uish both base residues and positions in the double-stranded DNA templ ate-primer stem.