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The role of MukE in assembling a functional MukBEF complex.

ABSTRACT: The MukB-MukE-MukF protein complex is essential for chromosome condensation and segregation in Escherichia coli. The central component of this complex, the MukB protein, is related functionally and structurally to the ubiquitous SMC (structural maintenance of chromosomes) proteins. In a manner similar to SMC, MukB requires the association of two accessory proteins (MukE and MukF) for its function. MukF is a constitutive dimer that bridges the interaction between MukB and MukE. While MukB can condense DNA on its own, it requires MukF and MukE to ensure proper chromosome segregation. Here, we present a novel structure of the E. coli MukE-MukF complex, in which the intricate crystal packing interactions reveal an alternative MukE dimerization interface spanning both N- and C-terminal winged-helix domains of the protein. The structure also unveils additional cross-linking interactions between adjacent MukE-MukF complexes mediated by MukE. A variant of MukE encompassing point mutations on one of these surfaces does not affect assembly of the MukB-MukE-MukF complex and yet cannot restore the temperature sensitivity of the mukE?kan strain, suggesting that this surface may mediate critical protein-protein interactions between MukB-MukE-MukF complexes. Since the dimerization interface of MukE overlaps with the region of the protein that interacts with MukB in the MukB-MukE-MukF complex, we suggest that competing MukB-MukE and MukE-MukE interactions may regulate the formation of higher-order structures of bacterial condensin.

SUBMITTER: Gloyd M

PROVIDER: S-EPMC3482342 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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The role of MukE in assembling a functional MukBEF complex.

Gloyd Melanie M Ghirlando Rodolfo R Guarné Alba A

Journal of molecular biology 20110810 4

The MukB-MukE-MukF protein complex is essential for chromosome condensation and segregation in Escherichia coli. The central component of this complex, the MukB protein, is related functionally and structurally to the ubiquitous SMC (structural maintenance of chromosomes) proteins. In a manner similar to SMC, MukB requires the association of two accessory proteins (MukE and MukF) for its function. MukF is a constitutive dimer that bridges the interaction between MukB and MukE. While MukB can con ...[more]

PMID: 21855551

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Project description:UnlabelledThe 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.ImportanceMechanistic understanding of how newly replicated bacterial chromosomes are segregated prior to cell division is incomplete. In this work, we provide in vivo experimental 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 the Escherichia coli chromosome by the SMC (structural maintenance of chromosome) complex, MukBEF. We provide in vivo data that support the demonstration in vitro that 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.

Dataset Information

The role of MukE in assembling a functional MukBEF complex.

Publications

The role of MukE in assembling a functional MukBEF complex.

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