Document 0711
 DOCN  M9590711
 TI    Efficient concerted integration of retrovirus-like dna by integrase from
       avian myeloblastosis virus and hiv-1.
 DT    9509
 AU    Grandgenett D; Vora A; McCord M; Goodarzi G; Im G; Brackmann K; St Louis
       University, St. Louis, MO
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Session I, speakers'
       abstracts - unpaged). Unique Identifier : AIDSLINE AIDS/95920008
 AB    We report the efficient concerted integration of a linear virus-like DNA
       donor into 2.8 Kbp circular DNA target by integrase (IN) purified from
       avian myeloblastosis virus (AMV) (Nucleic Acid Res. 22, 4454-4461,
       1994). The donor was 487 bp (M-2), 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 digestion and agarose gel
       electrophoresis. The donor also contained the SupF gene that was used
       for genetic selection of individual full-site recombinants to verify the
       host duplication size. Two different pathways, involving either one
       donor or two donor molecules, were used to produce full- site
       recombinants. About 95% 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 pairing 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. Under standard assay conditions (30 min at 37 degrees C),
       about 20 to 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. Supercoiled or nicked circular DNA could
       equally serve as target substrate. By modifying the standard assay
       conditions, we are now able to assemble AMV IN/donor nucleoprotein
       complexes that routinely produce concerted reaction products totaling
       50% of all recombinants. The same concerted integration events observed
       in the avian system also occurred using nonionic detergent-lysed HIV-1
       virions, Mg2+, and the appropriate HIV-1 donor molecule (H-2) that had
       the correct U3 and U5 LTR termini. Donor molecules possessing only U5
       termini also served as donor substrates. The HIV-1 integrase also
       preferred using two donor molecules for full-site events. Sequencing of
       individual full-site recombinants verified the presence of the HIV-1 5
       bp target site duplication.
 DE    DNA Nucleotidyltransferases/*METABOLISM  DNA,
       Viral/CHEMISTRY/*GENETICS/METABOLISM  Electrophoresis, Agar Gel  Genes,
       Reiterated  HIV-1/*ENZYMOLOGY/GENETICS  Myeloblastosis Virus,
       Avian/*ENZYMOLOGY/GENETICS  Nucleic Acid Conformation  Recombination,
       Genetic  Restriction Mapping  Virus Integration  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).