title: Enzymatic Capability of HIS-Tagged HIV-1 Integrase Using Oligonucleotide Disintegration Substrates.
abstract: Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression in Escherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6xHis tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8-7.2 in the presence of 2 mM MnCl(2). Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3' end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5' oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3' end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5'-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another. Copyright 1996 S. Karger AG, Basel
authors.affiliation: Department of Medicine, McGill University, Montreal, Que., Canada.
authors.forename: E.A.
authors.initials: EA
authors.lastname: Faust
authors.forename: A.
authors.initials: A
authors.lastname: Garg
authors.forename: L.
authors.initials: L
authors.lastname: Small
authors.forename: A.
authors.initials: A
authors.lastname: Acel
authors.forename: R.
authors.initials: R
authors.lastname: Wald
authors.forename: B.
authors.initials: B
authors.lastname: Udashkin