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

Abstract

A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a shortlived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The crosslinking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N 2 amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes. HIV-11 reverse transcriptase (RT) is a complex molecular machine that uses several kinetically distinct steps to incorporate a nucleotide into a growing DNA strand. It is a heterodimer composed of a larger 560-residue subunit (p66) and a smaller subunit (p51) that contains the N-terminal 440 residues of p66. Both subunits contain subdomains that were named fingers, palm, thumb, and connection, because of the similarity of p66 to a right hand. The DNA polymerase active site is located in the p66 palm subdomain and the DNA binding cleft is formed primarily by the p66 fingers, palm, and thumb subdomains. The mechanism of polymerization by RT is similar to other polymerases and involves: 1) binding of the DNA substrate to the apo-enzyme; 2) binding of dNTP and divalent metal ions (required for catalysis) to the enzyme⅐DNA complex, followed by rate-limiting conformational changes; 3) formation of a phosphodiester bond between the 3Ј-OH primer terminus and the ␣-phosphate of dNTP, followed by release of the pyrophosphate product; 4) translocation of the elongated DNA primer (for processive synthesis) from the dNTP-binding site (N site) to the priming site (P site) or release of the nucleic acid (distributive synthesis) (Fig. 1).Extensive biochemical and crystallographic studies have enhanced our understanding of the details of the mechanism of DNA polymerization. However, the translocation step rem...

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA)

“…Template and primer were first annealed at 95°C for 5 min, cooled at room temperature for 20 min, and stored at −20°C. The 15 μM annealed template/primer (T/P) substrates were incubated with 7 μM cross-linkable HIV RT in 25 mM Tris, pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , and 0.1 mM 2′,3′-dideoxyguanosine-5′-triphosphate (ddG-TP) (GE Health Life Science) to obtain the RT/DNA ddG-MP P complex; 70 μM EFdA-TP and 0.1 mM deoxythymidine monophosphate (dT-MP) for the RT/DNA EFdA-MP P •dT-MP N binary complex; or 70 μM EFdA-TP for the RT/DNA EFdA-MP P •EFdA-MP N and RT/DNA EFdA-MP P •EFdA-MP *N complexes at 31°C for 18 h. Cross-linked RT-dsDNA complex was purified using a Ni-Heparin tandem column strategy (20). Cross-linking efficiency was judged by an increase in the molecular weight of the p66 subunit in nonreducing SDS/PAGE.…”

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

“…The other major resistance mechanism involves ATP-mediated phosphorolytic excision of the incorporated chain-terminating NRTI from the 3Ј-end of the primer (27,28). We and others have previously shown that for excision to occur, the 3Ј-end of the primer must be positioned at the pre-translocation or N-site of RT (19,29,30). As we have already shown, the 3Ј-EFdA-MP-terminated primer strand binds predominantly in a pre-translocation mode.…”

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

“…The latter configuration is referred to as the post-translocational state. Cross-linking experiments revealed that pyrophosphorolysis or the ATP-dependent excision can only occur in the former pre-translocational state (26,27). To account for the non-competitive mode of inhibition it has been suggested that PFA may likewise bind to the pre-translocated complex (8).…”