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DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

ABSTRACT: We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein-protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (-)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.

SUBMITTER: Bigot S

PROVIDER: S-EPMC2875013 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

Bigot Sarah S Marians Kenneth J KJ

Nucleic acids research 20100116 9

We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(-)ve sc] DNA. The DNA translocation activity of FtsK was not required for stim ...[more]

PMID: 20081205

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Project description:UnlabelledBacterial FtsK plays a key role in coordinating cell division with the late stages of chromosome segregation. The N-terminal membrane-spanning domain of FtsK is required for cell division, whereas the C-terminal domain is a fast double-stranded DNA (dsDNA) translocase that brings the replication termination region of the chromosome to midcell, where it facilitates chromosome unlinking by activating XerCD-dif site-specific recombination. Therefore, FtsK coordinates the late stages of chromosome segregation with cell division. Although the translocase is known to act as a hexamer on DNA, it is unknown when and how hexamers form, as is the number of FtsK molecules in the cell and within the divisome. Using single-molecule live-cell imaging, we show that newborn Escherichia coli cells growing in minimal medium contain ~40 membrane-bound FtsK molecules that are largely monomeric; the numbers increase proportionately with cell growth. After recruitment to the midcell, FtsK is present only as hexamers. Hexamers are observed in all cells and form before any visible sign of cell constriction. An average of 7 FtsK hexamers per cell are present at midcell, with the N-terminal domain being able to hexamerize independently of the translocase. Detergent-solubilized and purified FtsK N-terminal domains readily form hexamers, as determined by in vitro biochemistry, thereby supporting the in vivo data. The hexameric state of the FtsK N-terminal domain at the division site may facilitate assembly of a functional C-terminal DNA translocase on chromosomal DNA.ImportanceIn the rod-shaped bacterium Escherichia coli, more than a dozen proteins act at the cell center to mediate cell division, which initiates while chromosome replication and segregation are under way. The protein FtsK coordinates cell division with the late stages of chromosome segregation. The N-terminal part of FtsK is membrane embedded and acts in division, while the C-terminal part forms a hexameric ring on chromosomal DNA, which the DNA can translocate rapidly to finalize chromosome segregation. Using quantitative live-cell imaging, which measures the position and number of FtsK molecules, we show that in all cells, FtsK hexamers form only at the cell center at the initiation of cell division. Furthermore, the FtsK N-terminal portion forms hexamers independently of the C-terminal translocase.

Dataset Information

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

Publications

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK.

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