Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases

Mol Cell Biol. 1992 May;12(5):2331-8. doi: 10.1128/mcb.12.5.2331-2338.1992.

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacteria / enzymology
  • Bacteria / genetics*
  • Base Sequence
  • DNA Nucleotidyltransferases / genetics*
  • DNA Nucleotidyltransferases / isolation & purification
  • DNA Nucleotidyltransferases / metabolism
  • DNA Transposable Elements*
  • Drosophila melanogaster / genetics
  • Integrases
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nucleotidyltransferases / genetics*
  • Nucleotidyltransferases / isolation & purification
  • Nucleotidyltransferases / metabolism
  • Oligodeoxyribonucleotides
  • Plasmids
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Recombination, Genetic
  • Retroviridae / enzymology
  • Retroviridae / genetics*
  • Schizosaccharomyces / genetics
  • Sequence Homology, Nucleic Acid
  • Transposases

Substances

  • DNA Transposable Elements
  • Oligodeoxyribonucleotides
  • Recombinant Fusion Proteins
  • DNA Nucleotidyltransferases
  • Integrases
  • Nucleotidyltransferases
  • Transposases