Cross-Linking of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Template-Primer

Abstract

Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used-photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N 2 -modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5 extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the "open" configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.Reverse transcriptase (RT) is the enzyme responsible for copying the single-stranded RNA genome of retroviruses into double-stranded DNA (dsDNA) (6,37,39). RT has two enzymatic activities: a polymerase that can copy either an RNA or a DNA template and an RNase H that can degrade RNA if (and only if) it is a part of an RNA-DNA hybrid. The RT of human immunodeficiency virus type 1 (HIV-1) is an important target for anti-HIV drugs; a number of the drugs approved for the treatment of HIV infections are RT inhibitors (27).HIV-1 RT is a heterodimer composed of two subunits: The larger, p66, is 560 amino acids long, and the smaller, p51, contains the first 440 residues of ...

Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

“…The side chains of amino acid residues of the polymerase active site D110, D185, and D186 of the p66 subunit from the ternary complex are shown. The superimposition was performed by using PROFIT program with five points fixed: two in the polymerase active site, residues 100 to 200 and 400 to 420 of p51, and the RNase H subdomain of p66 (29). The position of the extended template strand was modeled based on the distance constraints that are in agreement with the recent structural data (Tuske et al, unpublished) and the data of our cross-linking experiments.…”

“…We previously showed that the sitedirected photocrosslinking of the fingers subdomain of HIV-1 RT to an extended template can be used to monitor changes in the distance between particular positions on the surface of the protein and a nucleic acid substrate and that we could obtain information about changes in the flexibility of the enzyme (29). Photocrosslinking experiments in which the cross-linking agents were attached to specific positions (i.e., positions 65, 67, 70, and 74) in the ␤3-␤4 loop of the fingers subdomain of p66 and photocrosslinked to the single-stranded extension of the template showed that cross-linking was significantly reduced in ternary complexes compared to binary complexes (29). We show here that NNRTI binding causes increased cross-linking in experiments with diazirine reagents (especially with the diazirine reagent with the longer linker) and, for some positions in the ␤3-␤4 loop, NNRTI binding shifts the preferred sites of interaction with the template.…”

Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA

“…Oligonucleotide Synthesis-Fluorinated oligonucleotides were prepared essentially as described previously (13) on a 10-mol scale, utilizing the 4,4Јdimethoxytrityl protected phosphoramidite derived either from O 6 -(2-p-nitrophenethyl)-2-fluoro-2Ј-deoxyinosine (13,14) or the commercially available 4,4Јdimethoxytrityl phosphoramidite from O 6 -(trimethylsilylethyl)-2-fluoro-2Ј-deoxyinosine (15) (ChemGenes, Ashland, MA) as the precursor of the dG residue to be modified. A support-bound 27-mer oligonucleotide (precursor of *27-Tem-C2 and -C3, Table I) containing an O 6 -(2-p-nitrophenethyl)-2-fluoro-2Ј-deoxyinosine residue at the eleventh position from the 5Ј-end was allowed to react as described previously (13) with a 90-fold molar excess of either bis(3-aminopropyl)disulfide dihydrochloride (3-carbon tether) or commercially available cystamine dihydrochloride (2-carbon tether) in 500 l of water and 300 l of triethylamine for 16 h at room temperature, followed by deprotection (3 days, 60°C) with concentrated NH 4 OH containing 0.05 M tetrabutylammonium fluoride (deblocks O 6 ).…”

“…Although retention times varied depending on the amount of oligonucleotide and diamine injected, the desired oligonucleotide was well separated from multiple minor impurities, most of which eluted either much earlier or much later, as well as from bis(3-aminopropyl)disulfide (t R , 13 min). Thus, preliminary dialysis (13,14) to remove excess bis(3-aminopropyl)disulfide was unnecessary. The isolated yield of purified *20-Pri-C3 was 306 A 260 nm (ϳ1.5 mol).…”

Peptide scalpels for site-specific dissection of the DNA–protein interface

“…Secondly, nucleotide building blocks equipped with a reactive functionality (aldehyde, aziridine, alkylating moiety) can be used in automated DNA synthesis for the production of reactive oligonucleotides [38]. Additionally, various approaches for postsynthetic regioselective modification to introduce reactive functionalities into DNA have been described [39].…”