Assembly, translocation, and activation of XerCD- dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

Abstract

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.

FtsK translocation permits discrimination between an endogenous and an imported Xer/ dif recombination complex

“…In contrast, FtsK specifically stops on XerCD/dif complexes, which is likely a prerequisite for the activation of recombination (14)(15)(16)25). Because FtsK appears to inhibit the first steps of recombination between dif Ng and dif GGI , we tested its capacity to stop translocating when encountering XerCD Ng / dif GGI complexes.…”

“…The selection pressure on the XerD-binding site may then reside in the capacity of the dif site to assemble a complex able to stop FtsK, which would require a particular interaction of XerD with its binding site involving bases not directly involved in catalysis. Using single-molecule FRET, it has recently been shown that XerCD/dif complexes may adopt different conformations (25). The nucleotides modified within dif GGI may favor one of these conformations that would not be recognized by FtsK.…”

“…The stripping activity is proposed to have functional implications in releasing MatP/matS-mediated cohesion between the ter regions of the sister chromosome during cytokinesis in E. coli (10). Exceptions to the stripping effect are XerCD/dif complexes, whether they are synapsed or not, at which t-FtsKαβγ Ec stops and resides for a very short time, during which a single round of recombination can be induced (25). Our results suggest an additional role of the stripping activity in stabilizing IMEXs.…”

Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

“…Upon reaching XerCD-bound dif , whether in a synapsed or unsynapsed conformation, it resided briefly and then dissociated without reversal. FtsK C activated recombination when it met synapsed XerCD-dif complexes, and then dissociated before the completion of recombination by XerCD [May et al, 2015].…”

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

“…This DNA motor protein localizes to the bacterial cell division septum and contributes to segregating the sister chromosomes into the daughter cells by translocating towards their replication termini. On chromosome dimers, FtsK stops at the Xer-bound dif sites and activates recombination, triggering resolution of the dimers to monomers (Aussel et al, 2002; Grainge et al, 2011; May et al, 2015). Without FtsK, Xer- dif synaptic complexes are formed, but do not lead to final recombination products (Aussel et al, 2002; Diagne et al, 2014; Grainge et al, 2011; Zawadzki et al, 2013).…”