| Abstract: | The in vitro assembly process for forming nucleoprotein complexes containing linear retrovirus-like DNA and integrase (IN) was investigated. Solution conditions that allowed avian myeloblastosis virus IN to efficiently pair two separate linear DNA fragments (each 487 bp in length) containing 3' OH recessed long terminal repeat termini were established. Pairing of the viral termini by IN during preincubation on ice permitted these nucleoprotein complexes to catalyze the concerted insertion of the two termini into a circular DNA target (full-site reaction), mimicking the in vivo reaction. The three major solution determinants were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethylene glycol. The aprotic solvent dioxane (15%) was significantly better (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half-site integration events. Half-site reactions by IN involved the insertion of a single donor terminus into circular pGEM. Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent half-site reactions from occurring at high NaCl concentrations. Under optimal solution conditions, the concerted integration reaction was directly proportional to a sixfold range of IN. The complexes appeared not to turn over, and few half-site donor-donor molecules were produced. In the presence of 0.15 or 0.35 M NaCl, dioxane prevented efficient 3' OH trimming of a blunt-ended donor by IN, suggesting that the complexes formed by IN with blunt-ended donors were different from those formed with donors containing 3' OH recessed termini for strand transfer. The results suggest that IN alone was capable of protein-protein and protein-DNA interactions that efficiently promote the in vitro concerted integration reaction. |
