The SMC Complex MukBEF Recruits Topoisomerase IV to the Origin of Replication Region in Live Escherichia coli
ABSTRACTTheEscherichia colistructuralmaintenance ofchromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interactin vitrothrough a direct contact between the MukB dimerization hinge and the C-terminal domain of ParC, the catalytic subunit of TopoIV. The interaction stimulates catalysis by TopoIVin vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV toori, with fluorescent fusions of ParC and ParE both forming cellular foci that colocalize with those formed by MukBEF throughout the cell cycle and in cells unable to initiate DNA replication. Removal of MukBEF leads to loss of fluorescent ParC/ParE foci. In the absence of functional TopoIV, MukBEF forms multiple foci that are distributed uniformly throughout the nucleoid, whereas multiple catenatedoris cluster at midcell. Once functional TopoIV is restored, the decatenatedoris segregate to positions that are largely coincident with the MukBEF foci, thereby providing support for a mechanism by which MukBEF acts in chromosome segregation by positioning newly replicated and decatenatedoris. Additional evidence for such a mechanism comes from the observation that in TopoIV-positive (TopoIV+) cells, newly replicatedoris segregate rapidly to the positions of MukBEF foci. Taken together, the data implicate MukBEF as a key component of the DNA segregation process by acting in concert with TopoIV to promote decatenation and positioning of newly replicatedoris.IMPORTANCEMechanistic understanding of how newly replicated bacterial chromosomes are segregated prior to cell division is incomplete. In this work, we providein vivoexperimental support for the view that topoisomerase IV (TopoIV), which decatenates newly replicated sister duplexes as a prelude to successful segregation, is directed to the replication origin region of theEscherichia colichromosome by the SMC (structuralmaintenance ofchromosome) complex, MukBEF. We providein vivodata that support the demonstrationin vitrothat the MukB interaction with TopoIV stimulates catalysis by TopoIV. Finally, we show that MukBEF directs the normal positioning of sister origins after their replication and during their segregation. Overall, the data support models in which the coordinate and sequential action of TopoIV and MukBEF plays an important role during bacterial chromosome segregation.