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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SummaryRecurrent hemorrhage has been reported in humans as a result of acquired antibody inhibitors which interfere with the crosslinking of fibrin by factor XIII. One type of these inhibitors (Type III) prevents activated factor XIII from acting on fibrin. We have generated an anti- fibrin monoclonal antibody, called mAb 4A5, which binds to a peptide sequence at the carboxyl-terminus of human fibrinogen γ-chains. MAb 4A5 acts like a Type III inhibitor and prevents proper factor XHI-mediated crosslinking. Pre-incubation of fibrinogen or pooled human plasma with mAb 4A5, but not mAb D2 (specific for the carboxyl terminus of fibrin α-chains), resulted in clots which are soluble in either 5 M urea or 1% monochloroacetic acid. SDS-PAGE and immunoblotting analysis of these clots confirmed that mAb 4A5 inhibited γ-chain crosslinking in plasma clots and fibrin clots. Results from a factor XIII activity assay demonstrated that biotinylcadaverine cross-linking into fibrin by factor XIII could be inhibited by mAb 4A5 but not mAb D2, arguing that mAb 4A5 acted by binding the crosslinking site of factor XIII. Studies of the immunoreactivity of these mAbs with 12 different animal species showed that the γ-chain epitope recognized by mAb 4A5 was more conserved than the α-chain epitope recognized by mAb D2. The species fibrinogens, recognized by mAb 4A5 in binding assays, also showed impaired crosslinking when mAb 4A5 was present during the clotting reaction.

SummaryBy deriving an anti-peptide monoclonal antibody, mAb 7A4, we characterized the relatively unstudied carboxyl-terminal end of the α-chain of human factor XIII, the plasma transglutaminase. MAb 7A4 was directed against the last eight amino acids (Gln-Ile-Gln-Arg-Arg-Pro-Ser-Met) and bound with a dissociation constant of 3.4 × 10−8 M. In a solid assay format, mAb 7A4 bound equally well to factor XIII obtained from human plasma, platelets or placenta. However, in a solution-phase assay format, the epitope was largely unavailable but could be readily exposed by heat denaturation. Tmmunoblotting showed that this epitope is conserved among all species of plasma factor XIII tested except rabbit suggesting that the carboxyl-terminus might be an important structural element. Other competitive binding experiments with synthetic peptides as inhibitors pointed toward the final carboxyl-terminal amino acid, Met-731, as an immunochemically important determinant. This was used advantageously to confirm the finding that the caiboxyl-leiminal Mel-731 is largely absent from placental factor XIII (1) as compared to platelet or plasma factor XIII.