ASSEMBLY AND CATALYTIC PROPERTIES OF RETROVIRUS INTEGRASE DNA COMPLEXES CAPABLE OF EFFICIENTLY PERFORMING CONCERTED INTEGRATION

The in vitro assembly process for forming nucleoprotein complexes cont aining linear retrovirus-like DNA and integrase (IN) was investigated, Solution conditions that allowed avian myeloblastosis virus IN to eff iciently pair two separate linear DNA fragments (each 487 bp in length ) containing 3' OH recessed long terminal repeat termini were establis hed, Pairing of the viral termini by IN during preincubation on ice pe rmitted these nucleoprotein complexes to catalyze the concerted insert ion of the two termini into a circular DNA target (full-site reaction) , mimicking the in vivo reaction, The three major solution determinant s were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethy lene glycol, The aprotic solvent dioxane (15%) was significantly bette r (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half site integration events, Half-site react ions by IN involved the insertion of a single donor terminus into circ ular pGEM, Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent h alf-site reactions from occurring at high NaCl concentrations, Under o ptimal solution conditions, the concerted integration reaction was dir ectly proportional to a sixfold range of IN, The complexes appeared no t 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 comple xes formed by IN with blunt-ended donors were different from those for med 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 concert ed integration reaction.