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...

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Protonation of the tricyclic antidepressant drug trimipramine with maleic acid, methanesulfonic acid and hydrochloric acid was studied using 1H, 13C and 15N NMR spectroscopy at natural abundance. The effect of counter ions on the protonation was compared under identical conditions of solvent, concentration and temperature using homonuclear and heteronuclear one- and two-dimensional experiments. Differential protonation of the terminal tertiary amine nitrogen is determined from the indirect spin-spin couplings, chemical shifts, 13C relaxation data and variable-temperature experiments. In the maleate salt, only one of the acidic protons is involved in protonation, the other being associated with the anion moiety. 15N chemical shifts of the protonated nitrogens are nearly linearly related to the pK(a) of the constituent acid.