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Kulkosky J , Jones KS , Katz RA , Mack JPG , Skalka AM
Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases
Molecular and Cellular Biology. 1992 ;12(5) :2331-2338
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Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions. On the basis of similarities with enzymes that catalyze analogous reactions, we propose that the invariant D and E residues may participate in coordination of the metal cofactor (Mn2+ or Mg2+) required for the catalytic activities of IN. We further speculate that a metal-DNA complex may be necessary to position both LTR and target DNA substrates for nucleophilic attack during the cleavage and joining reactions.
02707306 (ISSN) Cited By: 264; Export Date: 31 May 2006; Source: Scopus CODEN: MCEBD Language of Original Document: English Correspondence Address: Skalka, A.M.; Fox Chase Cancer Center; Institute for Cancer Research Philadelphia, PA 19111, United States Chemicals/CAS: amino acid, 65072-01-7; aspartic acid, 56-84-8, 6899-03-2; glutamic acid, 11070-68-1, 138-15-8, 56-86-0, 6899-05-4, DNA Nucleotidyltransferases, EC 2.7.7.-; DNA Transposable Elements; Integrase, EC 2.7.7.-; Nucleotidyltransferases, EC 2.7.7; Oligodeoxyribonucleotides; Plasmids; Recombinant Fusion Proteins; Transposase, EC 2.7.7.-