Temporal coordination between initiation of HIV (+)-strand DNA synthesis and primer removal

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcri ptase (RT) using an in vitro system that closely mimics the initiation of ( +)-strand DNA synthesis, Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity st arts to cleave the RNA primer from newly synthesized DNA. Comparison of cle avage profiles obtained with 3'- and 5'-end-labeled primer strands indicate s that RT now translocates in the opposite direction, i.e. in the 5' direct ion of the RNA strand. DNA synthesis resumes again in the 3' direction, aft er the RNA-DNA junction was efficiently cleaved. Moreover, we further chara cterized complexes generated before, during, and after position +12, by tre ating these with Fe2+ to localize the RNase H active site on the DNA templa te. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus , whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA These data show that the initiation of (+)-strand sy nthesis is accompanied by a conformational change of the polymerase-compete nt complex.