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.Mutations and genetic rearrangements are frequently associated with repetitive DNA sequences (1). Genetic rearrangements are the deletion or expansion of repeated DNA sequences, leading to genomic changes that are important for bacterial survival or evolution (2). Transposon precise excision is a special case of deletion at short direct repeats created by the transposition mechanism and facilitated by the large inverted repeats constituting the insertion sequences of these elements (3). Precise and nearly precise excision are host-mediated processes that occur in the absence of recA function or any transposon-encoded functions (3). tex (for transposon excision) mutations have been identified that elevate the frequency of these rearrangements (4). The mutations include unusual recBC alleles that alter but do not abolish the functions of RecBC (5) and alleles of genes involved in the methylation-directed pathway for repair of base pair mismatches such as mutS, mutH, mutL, dam, and uvrD (6). Conditional mutations in ssb, the essential gene encoding the Escherichia coli single-stranded DNA-binding protein SSB, also confer a tex phenotype (6, 7). Recently it was reported that mutations in topA (toposimerase I), dnaA (DNA polymerase I), and dnaBE (helicase and ␣-subunits of DNA polymerase III) also lead to the same phenotype (8, 9). All of these mutations affect genes encoding proteins involved in DNA replication or repair, supporting the notion that deletions are the result of errors during replication (1).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). Molecular cloning and nucleotide sequence determination of the uup gene suggested that the Uup ...