The SMC Complex MukBEF Recruits Topoisomerase IV to the Origin of Replication Region in Live Escherichia coli

Abstract

The Escherichia coli structural maintenance of chromosome (SMC) complex, MukBEF, and topoisomerase IV (TopoIV) interact in vitro through 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 TopoIV in vitro. Using live-cell quantitative imaging, we show that MukBEF directs TopoIV to ori, 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 catenated oris cluster at midcell. Once functional TopoIV is restored, the decatenated oris 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 decatenated oris. Additional evidence for such a mechanism comes from the observation that in TopoIV-positive (TopoIV+) cells, newly replicated oris 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 replicated oris.

The bacterial nucleoid: nature, dynamics and sister segregation

“…Colocalization of sister loci behind the fork is usually thought to involve “precatenanes”, topological intertwining between sisters that would be driven by alleviation of the replication “swivel problem” [34–37]. Catenated sisters have been observed directly on small plasmids and linking of sisters by precatenanes has been inferred for the main chromosome from the fact that absence of TopoIV results in aberrant segregation and delayed locus splitting [27,35,38].…”

Bacterial Chromosome Organization and Segregation

“…Additionally, a major challenge is to determine whether MukBEF promotes chromosome segregation indirectly by condensing DNA, which may make other mechanisms of segregation operate more efficiently, or whether the MukBEF complex plays a more active role in directly partitioning sister chromosomes. Recent studies have demonstrated that MukBEF directly stimulates Topo IV, implying that MukBEF contributes to the disentangling of sister chromosomes (Hayama & Marians 2010, Li et al 2010, Nicolas et al 2014). …”

MukBEF, a Chromosomal Organizer

“…A weak interaction between MukB and ParC was recently discovered using co-immunoprecipitation [Hayama and Marians, 2010; Li et al, 2010b]. This interaction is sufficiently selective to discriminate between topo IV and DNA gyrase [Vos et al, 2013] and sufficiently strong to recruit a subset of topo IVs to the quarter positions inside the cell [Nicolas et al, 2014]. Mutations that disrupt MukB-ParC interface adversely affect such localization and E. coli viability [Hayama and Marians, 2010; Li et al, 2010b].…”