Development of an acid-soluble assay for measuring retrovirus integrase 3′-OH terminal nuclease activity

Abstract

A quantitative and efficient assay was developed to measure the 3′-OH terminal DNA endonuclease activity of the avian myeloblastosis virus (AMV) integrase protein. A retroviral-like linearized plasmid containing long terminal repeat (LTR) sequences at its recessed 3′-OH termini was filled in and labeled with the Escherichia coli Klenow DNA polymerase fragment. The 32P-labeled nucleotide was located at the penultimate position. The labeled linearized plasmid or restriction fragments derived from it were incubated with AMV IN and release of the label was quantitated by conversion to acid-soluble counts. The structure of the released product was characterized on 23% sequencing gels. Results indicate that AMV integration protein is functioning as an endonuclease releasing a dinucleotide and that the activity is stoichiometric with a preference for the cleavage of the U3 LTR terminus over that of the U5 LTR terminus.