Reverse transcriptase (RT)-2؉ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg 2؉ -dependent cleavage. The Fe 2؉ -dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3-end and the Fe 2؉ -dependent cleavage position at the RNase H active site. Retroviral reverse transcriptases (RTs)1 are multifunctional enzymes having an RNA-dependent and a DNA-dependent polymerase activity and a ribonuclease H (RNase H) activity, which degrades the RNA strand of RNA/DNA hybrids (1). These activities facilitate the conversion of single-stranded genomic RNA to the double-stranded proviral DNA which is integrated into the host genome. Synthesis of the first DNA strand, the minus strand DNA, is initiated from a cellular tRNA which binds with its 3Ј terminus to the complementary primer-binding site near the 5Ј-end of the viral RNA (2-4). The newly synthesized minus-strand DNA serves in turn as template for the synthesis of the second DNA strand or plusstrand. Prerequisite for the plus-strand synthesis is removal of the viral RNA from the minus-strand DNA. Both activities act simultaneously, presupposing a spatial and temporal interdependence of the active sites.The interplay of the two activities has been studied extensively in RT of human immunodeficiency virus type 1 (HIV-1) (5-13). It has been demonstrated that the viral RNA template is cleaved 18 nucleotides upstream with respect to the 3Ј-end of the nascent primer terminus (7) during minus-strand synthesis. But cleavage does not occur after each nucleotide incorporation step, indicating that the two activities, the polymerase and RNase H activity, are not coupled in a 1:1 mode (11-14). Gopalakrishnan et al. (7) suggested a temporal coordination of the two activities due to kinetic coupling. Cleavage can occur if the rate of DNA polymerization is lower than the rate of RNA hydrolysis. A similar concept might also explain the resistance of dsRNA toward cleavage. It has been shown that dsRNA formed by the tRNA and the viral RNA during initiation of minus-strand synthesis remains intact during DNA synthesis, but is cleaved, if the DNA synthesis is artificially stopped through use of chain terminating nucleotides (9). This can be explained by the fact that the rate of polymerization is higher than the rate of RNA template hydrolysis of dsRNA in line with the kinetic coupling model (9). Ribonuclease activity on dsRNA, which is believed to be mediated by t...

In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (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 starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3-and 5-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5 direction of the RNA strand. DNA synthesis resumes again in the 3 direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position ؉12, by treating these with Fe 2؉ to localize the RNase H active site on the DNA template. 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 synthesis is accompanied by a conformational change of the polymerase-competent complex. Retroviral RTs1 are multifunctional enzymes possessing RNA-and DNA-dependent polymerase activities and a ribonuclease H (RNase H) activity that degrades the RNA strand of RNA/DNA hybrids (1, 2). Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) uses a cellular tRNA primer to initiate reverse transcription from a complementary primer-binding site (PBS) near the 5Ј-end of the viral RNA (3-6). Despite changes of binding and kinetic properties, observed concomitant with synthesis of the first DNA strand (7), i.e. (Ϫ)-strand DNA, complexes with the initially bound RNA/ RNA duplex and the newly synthesized DNA/RNA substrates share certain common features. RNase H cleavages on the RNA strand of DNA/RNA primer/template combinations occur at a constant distance of 18 bp upstream of the nascent primer terminus (8, 9). Analogously, RNase H-induced cleavages within the tRNA/RNA duplex, designated as RNase H* activity (10), were observed at the same distance from the 3Ј-end of the primer, although these cuts are restricted to stalled complexes (11). Together, these data provide strong evidence that RT binds to both RNA/RNA and DNA/RNA substrates with the same orientation, and the number of bp between the two active sites is 18 in each case.RT-DNA/DNA complexes, which are generated during (ϩ)-strand synthesis, have been relatively well characterized (12-15). The crystal structure of HIV-1 RT complexed to an 18-base primer/19-base template DNA homoduplex (12) suggests that the first 7 DNA/DNA base pairs near the polymerase active site adopt an A-type conformation, whereas the region further upstream is in the preferred B-conformation, both structurally distinct segments being separated by a kink.Little information is currently available regarding th...

During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3'-DNA-RNA-5' junction is formed involving the 3'-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 A resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding Keywords: HIV-1 reverse transcriptase; heterologous expression; GST-fusion; coexpression; helix-clamp motif.The reverse transcriptase (RT) of HIV-1, the causative agent of AIDS, catalyzes the synthesis of the proviral dsDNA, a crucial step in viral replication [1,2]. Replication starts with the synthesis of minus-strand DNA initiated from the 3 H -end of the host cellular tRNA Lys3 , which is complementary to a sequence of the viral ssRNA. The synthesis of proviral dsDNA therefore requires an RNA-dependent polymerase activity as well as an RNase H activity to degrade the copied genomic RNA template. Initation of plus-strand DNA synthesis from the so-called polypurine tract requires a DNA-dependent polymerase activity. Thus, during replication the RT needs to bind diverse primer/template substrates such as dsRNA, RNA/DNA, DNA/RNA and dsDNA.RT consists of two subunits, where the smaller p51 subunit is generated from C-terminal proteolytic cleavage of the larger p66 subunit by viral protease [3]. Although both subunits harbour the same N-terminus, the crystal structure reveals an asymmetric heterodimer in which p66 comprises the polymerase as well as the RNase H active sites [4,5]. It has been suggested that a helixturn-helix structure, termed the helix clamp, is involved in nucleic acid binding in both subunits [6].The aim of this work is to establish a system for obtaining mixed reconstituted RT which consists of selectively mutated subunits in order to analyse t...