title: Viral long terminal repeat substrate binding characteristics of the human immunodeficiency virus type 1 integrase.
abstract: A DNA binding assay was developed for the human immunodeficiency virus type 1 (HIV-1) integrase. The assay was capable of defining discrete complexes between the enzyme and the viral long terminal repeat (LTR) substrate. DNA binding reflected the sequence requirements previously demonstrated for the enzyme's 3'-end processing activity. Binding exhibited a nonlinear dependence on integrase concentration, suggesting that the enzyme functions as a multimer. The oligomeric state was investigated by UV-photo-cross-linking of integrase-LTR oligonucleotide complexes using DNA substrates substituted with 5-bromo-2'-deoxycytidine within the integrase recognition sequence. In the absence of divalent cation, integrase cross-linked to the LTR oligonucleotide as a single species whose mobility by SDS-polyacrylamide gel electrophoresis was consistent with the formation of tetramers. Using these techniques, analysis of the binding properties of integrase mutants demonstrated that the catalytic and sequence-specific DNA binding activities of the enzyme are distinct, involving residues within the conserved "DD(35)E" and zinc finger motifs, respectively.
authors.affiliation: Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: D J
authors.initials: DJ
authors.lastname: Hazuda
authors.forename: A L
authors.initials: AL
authors.lastname: Wolfe
authors.forename: J C
authors.initials: JC
authors.lastname: Hastings
authors.forename: H L
authors.initials: HL
authors.lastname: Robbins
authors.forename: P L
authors.initials: PL
authors.lastname: Graham
authors.forename: R L
authors.initials: RL
authors.lastname: LaFemina
authors.forename: E A
authors.initials: EA
authors.lastname: Emini
mesh_terms: D001483:Base Sequence; D002413:Cations, Divalent; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology*; D019426:Integrases; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016335:Zinc Fingers
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases