TnpA(REP) and REP sequences dissemination in bacterial genomes: REP recognition determinants

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpA(REP), the first prokaryotic domesticated transposase associated with REP. TnpA(REP), fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpA(REP) processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpA(REP). To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpA(REP)/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpA(REP) groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.