Abstract
The U3 and U5 termini of linear retrovirus DNA contain imperfect inverted repeats that are necessary for the concerted insertion of the termini into the host chromosome by viral integrase. Avian myeloblastosis virus integrase can efficiently insert the termini of retrovirus-like DNA donor substrates (480 base pairs) by a concerted mechanism (full-site reaction) into circular target DNA in vitro. The specific activities of virion-derived avian myeloblastosis virus integrase and bacterial recombinant Rous sarcoma virus (Prague A strain) integrase (approximately 50 nM or less) appear similar upon catalyzing the full-site reaction with 3'-OH recessed wild type or mutant donor substrates. We examined the role of the three nonsymmetrical nucleotides located at the 5th, 8th, and 12th positions in the U3 and U5 15-base pair inverted repeats for their ability to modify the full-site and simultaneously, the half-site strand transfer reactions. Our data suggest that the nucleotide at the 5th position appears to be responsible for the 3-5-fold preference for wild type U3 ends over wild type U5 ends by integrase for concerted integration. Additional mutations at the 5th or 6th position, or both, of U3 or U5 termini significantly increased (approximately 3 fold) the full-site reactions of mutant donors over wild type donors.
Highlights
Mutagenesis Strategy Used to Investigate the Full-site Integration Reaction in Vitro—In this report we examined the contribution of the three nonsymmetrical nucleotides that map to the 5th, 8th, and 12th positions of the RSV U3 and U5 LTR DNA termini for the full-site integration reaction (Fig. 1)
A wt LTR terminus was present on the opposite end of each LTR mutant donor substrate
To determine if sequences downstream of the 8th nonsymmetrical nucleotide of U3 were important, we investigated whether a single nucleotide deletion (A at position 10) in the U3 LTR of donor U5P8-G/A had an effect on catalysis
Summary
Vol 272, No 38, Issue of September 19, pp. 23938 –23945, 1997 Printed in U.S.A. ROLE OF NONSYMMETRICAL NUCLEOTIDES IN PROMOTING FULL-SITE INTEGRATION BY PURIFIED VIRION AND BACTERIAL RECOMBINANT INTEGRASES*. The half-site reaction involves the insertion of only one LTR terminus into the DNA target These in vitro analyses using purified integrase from several retrovirus species have established that the imperfect inverted repeat sequences located at the LTR termini are necessary for catalysis [1, 11, 12]. In this report we examined the role of the three nonsymmetrical nucleotides located in the avian U3 and U5 15-bp inverted repeat and several other LTR mutations in the formation of donor-target recombinants that are produced by the full-site integration reaction. The specific activities of both virion and recombinant integrase appear equivalent when catalyzing the half-site and full-site strand transfer reactions with either wt or mutant LTR DNA sub-. Strates, or the 3Ј-OH processing reaction with similar blunt ended substrates
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