ATP Hydrolysis Is Essential for the Function of the Uup ATP-binding Cassette ATPase in Precise Excision of Transposons

Pierre Bance,Dorothée Murat,Elie Dassa,Isabelle Callebaut

doi:10.1074/jbc.m509926200

Abstract

In Escherichia coli K-12, the RecA- and transposase-independent precise excision of transposons is thought to be mediated by the slippage of the DNA polymerase between the two short direct repeats that flank the transposon. Inactivation of the uup gene, encoding an ATP-binding cassette (ABC) ATPase, led to an important increase in the frequency of precise excision of transposons Tn10 and Tn5 and a defective growth of bacteriophage Mu. To provide insight into the mechanism of Uup in transposon excision, we purified this protein, and we demonstrated that it is a cytosolic ABC protein. Purified recombinant Uup binds and hydrolyzes ATP and undergoes a large conformational change in the presence of this nucleotide. This change affects a carboxyl-terminal domain of the protein that displays predicted structural homology with the socalled little finger domain of Y family DNA polymerases. In these enzymes, this domain is involved in DNA binding and in the processivity of replication. We show that Uup binds to DNA and that this binding is in part dependent on its carboxyl-terminal domain. Analysis of Walker motif B mutants suggests that ATP hydrolysis at the two ABC domains is strictly coordinated and is essential for the function of Uup in vivo.

Highlights

Mutations in topA, dnaA (DNA polymerase I), and dnaBE lead to the same phenotype [8, 9]
Molecular cloning and nucleotide sequence determination of the uup gene suggested that the Uup protein is cytosolic and belongs to the superfamily of ATP-binding cassette (ABC)3 proteins [7, 9]. tex mutations affecting transposon precise excision fall into two categories: the first, such as uup, ssb, topA, and polA, increase precise excision of both Tn10 and mini-Tn10; and the second, such as mutHLS, dam, and uvrD, increase precise excision of Tn10 but not of mini-Tn10
All ABC proteins are characterized by the presence of a conserved domain, known as the nucleotide-binding domain (NBD), which contains at least five characteristic sequence motifs [12]

Summary

Introduction

Mutations in topA (toposimerase I), dnaA (DNA polymerase I), and dnaBE (helicase and ␣-subunits of DNA polymerase III) lead to the same phenotype [8, 9]. Other tex mutations affect the uup gene and cause an increase in the frequency of precise excision of transposons Tn10, mini-Tn10, and Tn5 and a defective growth of bacteriophage Mu [10]. ABC proteins constitute one of the largest families of paralogues in sequenced genomes [11] They couple ATP hydrolysis to a wide variety of cellular processes, including transmembrane transport, gene regulation, and DNA repair. We demonstrate that recombinant Uup has intrinsic ATPase activity and interacts with DNA in vitro This binding is in part dependent on the 90-amino acid carboxyl-terminal domain of Uup that shares similarities with little finger domains found in the Y family of DNA polymerases. Analysis of Walker motif B mutants led to the conclusion that ATP hydrolysis at the two ABC domains of the protein is essential for the function of Uup in vivo

Methods

Results

Conclusion