EFFICIENT CONCERTED INTEGRATION OF RETROVIRUS-LIKE DNA IN-VITRO BY AVIAN-MYELOBLASTOSIS VIRUS INTEGRASE

We report the efficient concerted integration of a linear virus-like D NA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained rece ssed 3' OH ends, was 5' end labeled, and had a unique restriction site not found in the target. Analysis of concerted (full-site) and half-s ite integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF ge ne that was used for genetic selection of individual full-site recombi nants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produc e full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imp ly that juxtapositioning an end from each of two donors by IN was more efficient than the juxtapositioning of two ends of a single donor for the full-site reaction. The formation of preintegration complexes con taining integrase and donor on ice prior to the addition of target enh anced the full-site reaction. After a 30 min reaction at 37 degrees C, similar to 20 - 25% of all donor/target recombinants were the result of concerted integration events. The efficient production of full-site recombinants required Mg2+; Mn2+ was only efficient for the productio n of half-site recombinants. We suggest that these preintegration comp lexes can be used to investigate the relationships between the 3' OH t rimming and strand transfer reactions.