The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits a marked preference for Mn(II) rather than Mg(II).
In this investigation, we examine the interaction between the human immunodeficiency virus type I integrase and oligonucleotides that reflect the sequences of the extreme termini of the viral long terminal repeats (LTRs). The results of gel filtration and a detailed binding density analysis indicate that the integrase binds to the LTR as a high-order oligomer at a density equivalent to 10 +/- 0.8 integrase monomers per 21-base pair LTR. The corresponding binding isotherm displays a Hill coefficient of 2, suggesting that the binding mechanism involves the cooperative interaction between two oligomers. This interaction is quite stable, exhibiting a prolonged half-life (t1/2 approximately 13 h) in the presence of Mn2+ cations. Complexes were less stable when formed with Mg2+ (t1/2 approximately 1 h). The role of Mn2+ appears to be in the induction of the protein-protein interactions that stabilize the bound complexes. In terms of the 3'-end processing of the LTR, similar catalytic rates (kcat approximately 0.06 min-1) were obtained for the stable complex in the presence of either cation. Hence, the apparent preference observed for Mn2+ in standard in vitro integration assays can be attributed entirely to the augmentation in the DNA binding affinity of the integrase.
Pemberton
I K
IK
Unité de Physicochimie des Macromolécules Biologiques CNRS URA 1149, Institut Pasteur, Paris, France.
Buckle
M
M
Buc
H
H
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019426:Integrases; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D016662:Virus Integration
D002413:Cations, Divalent; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D008274:Magnesium