THE ROLE OF MANGANESE IN PROMOTING MULTIMERIZATION AND ASSEMBLY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE AS A CATALYTICALLY ACTIVE COMPLEX ON IMMOBILIZED LONG TERMINAL REPEAT SUBSTRATES

The integration of a DNA copy of the viral genome into the genome of t he host cell is an essential step in the replication of all retrovirus es, Integration requires two discrete biochemical reactions: specific processing of each viral long terminal repeat terminus or donor substr ate, and a DNA strand transfer step wherein the processed donor substr ate is joined to a nonspecific target DNA, Both reactions are catalyze d by a virally encoded enzyme, integrase. A microtiter assay for the s trand transfer activity of human immunodeficiency virus type 1 integra se which uses an immobilized oligonucleotide as the donor substrate wa s previously published (D, J, Hazuda, J. C, Hastings, A, L. Wolfe, and E, A, Emini, Nucleic Acids Res. 22:1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA, The enzyme which binds to the immobilized donor is show n to be sufficient to catalyze strand transfer with target DNA substra tes added subsequent to assembly; in the absence of the target substra te, the complex was retained on the donor in an enzymatically competen t state, Assembly required high concentrations of divalent cation, wit h optimal activity achieved at 25 mM MnCl2, In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the di valent cation in assembly and catalysis, respectively, Prior incubatio n of the enzyme in 25 mM MnCl2 was shown to promote the multimerizatio n of integrase in the absence of a DNA substrate and alleviate the req uirement for high concentrations of divalent cation during assembly, T he superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro, Subunits were ob served to exchange during the assembly reaction, suggesting that multi merization can occur either before or coincident with but not after do nor binding, These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.