The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products

Robert J. Sarnovsky, Earl W. May, Nancy L Craig

Research output: Contribution to journalArticle

Abstract

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB; also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.

Original languageEnglish (US)
Pages (from-to)6348-6361
Number of pages14
JournalThe EMBO journal
Volume15
Issue number22
StatePublished - Nov 15 1996

Fingerprint

Transposases
Joining
Genes
Metals
DNA
Catalytic Domain
Acidic Amino Acids
Integrases
DNA Cleavage
Recombinases
Proteins
Ointments
Site-Directed Mutagenesis
Sulfur
Mutagenesis
Cysteine
Ions
Metal ions
Catalyst activity
Mutation

Keywords

  • Active site
  • DD(35)E motif
  • DNA transposition
  • Protein-DNA complex
  • Transposase

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. / Sarnovsky, Robert J.; May, Earl W.; Craig, Nancy L.

In: The EMBO journal, Vol. 15, No. 22, 15.11.1996, p. 6348-6361.

Research output: Contribution to journalArticle

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