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 DNA donor into a 2.8 kbp circular DNA target by integrase (IN) purified from avian myeloblastosis virus. The donor was 528 bp, contained recessed 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-site integration events was accomplished by restriction enzyme analysis and agarose gel electrophoresis. The donor also contained the SupF gene that was used for genetic selection of individual full-site recombinants to determine the host duplication size. Two different pathways, involving either one donor or two donor molecules, were used to produce full-site recombinants. About 90% of the full-site recombinants were the result of using two donor molecules per target. These results imply 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 containing integrase and donor on ice prior to the addition of target enhanced the full-site reaction. After a 30 min reaction at 37 degrees C, approximately 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 production of half-site recombinants. We suggest that these preintegration complexes can be used to investigate the relationships between the 3' OH trimming and strand transfer reactions.
Vora
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AC
Institute for Molecular Virology, St Louis University Health Sciences Center, MO 63110.
McCord
M
M
Fitzgerald
M L
ML
Inman
R B
RB
Grandgenett
D P
DP
D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001426:Bacterial Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D019426:Integrases; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology
D001426:Bacterial Proteins; D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D004262:DNA Restriction Enzymes; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium