Grandgenett D, Vora A, McCord M, Goodarzi G, Im G, Brackmann K; NIH Conference on Retroviral Integrase.

NIH Conf Retroviral Integr NIH Conf Retroviral Integr 1995 Bethesda Md. 1995 Jan 19-20; (Session I, speakers' abstracts - unpaged).

St Louis University, St. Louis, MO

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.

Publication Types:

• Meeting Abstracts

Keywords:

• Avian myeloblastosis virus
• DNA
• DNA Footprinting
• DNA Nucleotidyltransferases
• DNA Restriction Enzymes
• DNA, Circular
• DNA, Viral
• Electrophoresis, Agar Gel
• HIV Integrase
• HIV-1
• Integrases
• Retroviridae
• Terminal Repeat Sequences
• Virion
• genetics

Other ID:

• 95920008

UI: 102212953

From Meeting Abstracts