IN-VIVO CLEAVAGE OF ESCHERICHIA-COLI BIME-2 REPEATS BY DNA GYRASE - GENETIC-CHARACTERIZATION OF THE TARGET AND IDENTIFICATION OF THE CUT SITE

The Escherichia coil chromosome contains about 300 bacterial intersper sed mosaic elements (BIMEs), These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extrageni c palindromes (REPs). Based on the type of REP they contain and on the ir ability to interact with the integration host factor (IHF), BIMEs a re subdivided into two families: BIME-1 elements contain an IHF bindin g site flanked by converging Y and Z1 REPs, whereas BIME-2 elements co ntain a variable number of alternating Y and Z2 REPs without an IHF si te, Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belo nging to both families remains to be elucidated, In this paper, we use d oxolinic acid, a drug that reveals potential sites of DNA gyrase act ion, to demonstrate that DNA gyrase interacts in vivo with BIME-5 elem ents, The frequency of cleavage varied from one element to another, an d the cleavage pattern observed in elements containing several REPs in dicated that DNA gyrase cut DNA every two REPs, A single cleavage site has been identified in the Y REP in six out of seven instances, and t he nucleotide sequence of a 44 bp fragment containing the scission poi nt displayed conserved residues at six positions. The lack of one of t he conserved residues accounted for the absence of cleavage in most of the 22 REPs. Our results also showed that cleaved REPs were always as sociated with another REP, suggesting that a pair of diverging REPs co nstitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rea rrangements.