Murat Dorothée, Bance Pierre, Callebaut Isabelle, Dassa Elie
Unité des Membranes Bactériennes CNRS URA2172, Département de Microbiologie Fondamentale et Médicale, Site Fernbach, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
J Biol Chem. 2006 Mar 10;281(10):6850-9. doi: 10.1074/jbc.M509926200. Epub 2006 Jan 5.
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.
在大肠杆菌K-12中，转座子不依赖RecA和转座酶的精确切除被认为是由DNA聚合酶在转座子两侧的两个短同向重复序列之间滑动介导的。编码ATP结合盒（ABC）ATP酶的uup基因失活导致转座子Tn10和Tn5精确切除频率显著增加，以及噬菌体Mu生长缺陷。为深入了解Uup在转座子切除中的机制，我们纯化了该蛋白，并证明它是一种胞质ABC蛋白。纯化的重组Uup结合并水解ATP，且在该核苷酸存在时发生大的构象变化。这种变化影响该蛋白的羧基末端结构域，该结构域与Y家族DNA聚合酶的所谓小手指结构域具有预测的结构同源性。在这些酶中，该结构域参与DNA结合和复制的持续性。我们表明Uup与DNA结合，且这种结合部分依赖于其羧基末端结构域。对沃克基序B突变体的分析表明，两个ABC结构域的ATP水解严格协调，并且对Uup在体内的功能至关重要。
