Dataset Information

The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF.

ABSTRACT: The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that are preferentially localized to the replication origin region (ori), close to the long axis of the cell. A replication-dependent increase in the fraction of immobile molecules, together with a proposed catalytic cycle of ~1.8 s, is consistent with the majority of active TopoIV molecules catalyzing decatenation, with a minority maintaining steady-state DNA supercoiling. Finally, we show that the MukB-ParC interaction is crucial for timely decatenation and segregation of newly replicated ori DNA.

SUBMITTER: Zawadzki P

PROVIDER: S-EPMC5061553 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF.

Zawadzki Pawel P Stracy Mathew M Ginda Katarzyna K Zawadzka Katarzyna K Lesterlin Christian C Kapanidis Achillefs N AN Sherratt David J DJ

Cell reports 20151210 11

The type II topoisomerase TopoIV, which has an essential role in Escherichia coli chromosome decatenation, interacts with MukBEF, an SMC (structural maintenance of chromosomes) complex that acts in chromosome segregation. We have characterized the intracellular dynamics of individual TopoIV molecules and the consequences of their interaction with MukBEF clusters by using photoactivated-localization microscopy. We show that ~15 TopoIV molecules per cell are associated with MukBEF clusters that ar ...[more]

PMID: 26686641

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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 Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF.

Publications

The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF.

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