Molecular mechanism of sequence-directed DNA loading and translocation by FtsK

Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.     

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.     

KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK γ regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region ( ter ) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif , located within ter . The frequency of chromosome dimer formation is ∼15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of ∼70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre– loxP recombination replaces XerCD– dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter .     

Extent of the activity domain and possible roles of FtsK in the Escherichia coli chromosome terminus

Escherichia coli FtsK protein couples cell division and chromosome segregation. It is a component of the septum essential for cell division. It also acts during chromosome dimer resolution by XerCD-specific recombination at the dif site, with two distinct activities: DNA translocation oriented by skewed sequence elements and direct activation of Xer recombination. Dimer resolution requires that the skewed elements polarize in opposite directions 30-50 kb on either side of dif. This constitutes the DIF domain, approximately coincident with the region where replication terminates. The observation that the ftsK1 mutation increases recombination near dif was exploited to determine whether the chromosome region on which FtsK acts is limited to the DIF domain. A monitoring of recombination activity at multiple loci in a 350 kb region to the left of dif revealed (i) zones of differing activities unconnected to dimer resolution and (ii) a constant 10-fold increase of recombination in the 250 kb region adjacent to dif in the ftsK1 mutant. The latter effect allows definition of an FTSK domain whose total size is at least fourfold that of the DIF domain. Additional analyses revealed that FtsK activity responds to polarization in the whole FTSK domain and that displacement of the region where replication terminates preserves differences between recombination zones. Our interpretation is that translocation by FtsK occurs mostly on DNA belonging to a specifically organized domain of the chromosome, when physical links between either dimeric or still intercatenated chromosomes force this DNA to run across the septum at division.     

Evidence from terminal recombination gradients that FtsK uses replichore polarity to control chromosome terminus positioning at division in Escherichia coli

Chromosome dimers in Escherichia coli are resolved at the dif locus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein. Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires the dif resolution site to be located at the junction between the two polarized chromosome arms or replichores. Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination). We had proposed that dif sites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes. To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in an ftsK mutant. The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on a xerC mutant chromosome with respect to intensity, the region affected, and response to polarity distortion. The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localize dif at the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material.     

Asymmetric activation of Xer site-specific recombination by FtsK

Chromosome dimers, which frequently form in Escherichia coli, are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific site on the chromosome, dif, together with the cell division protein FtsK. The C-terminal domain of FtsK (FtsK(C)) is a DNA translocase implicated in helping synapsis of the dif sites and in locally promoting XerD strand exchanges after synapse formation. Here we show that FtsK(C) ATPase activity is directly involved in the local activation of Xer recombination at dif, by using an intermolecular recombination assay that prevents significant DNA translocation, and we confirm that FtsK acts before Holliday junction formation. We show that activation only occurs with a DNA segment adjacent to the XerD-binding site of dif. Only one such DNA extension is required. Taken together, our data suggest that FtsK needs to contact the XerD recombinase to switch its activity on using ATP hydrolysis.     

Staphylococcus aureus requires at least one FtsK/SpoIIIE protein for correct chromosome segregation

Faithful coordination between bacterial cell division and chromosome segregation in rod‐shaped bacteria, such as Escherichia coli and Bacillus subtilis, is dependent on the DNA translocase activity of FtsK/SpoIIIE proteins, which move DNA away from the division site before cytokinesis is completed. However, the role of these proteins in chromosome partitioning has not been well studied in spherical bacteria. Here, it was shown that the two Staphylococcus aureus FtsK/SpoIIIE homologues, SpoIIIE and FtsK, operate in independent pathways to ensure correct chromosome management during cell division. SpoIIIE forms foci at the centre of the closing septum in at least 50% of the cells that are close to complete septum synthesis. FtsK is a multifunctional septal protein with a C‐terminal DNA translocase domain that is not required for correct chromosome management in the presence of SpoIIIE. However, lack of both SpoIIIE and FtsK causes severe nucleoid segregation and morphological defects, showing that the two proteins have partially redundant roles in S. aureus.     

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.     

FtsK translocation on DNA stops at XerCD-dif

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsKγ regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.     

The Bacillus subtilis SftA (YtpS) and SpoIIIE DNA translocases play distinct roles in growing cells to ensure faithful chromosome partitioning

In several bacterial species, the faithful completion of chromosome partitioning is known to be promoted by a conserved family of DNA translocases that includes Escherichia coli FtsK and Bacillus subtilis SpoIIIE. FtsK localizes at nascent division sites during every cell cycle and stimulates chromosome decatenation and the resolution of chromosome dimers formed by recA -dependent homologous recombination. In contrast, SpoIIIE localizes at sites where cells have divided and trapped chromosomal DNA in the membrane, which happens during spore development and under some conditions when DNA replication is perturbed. SpoIIIE completes chromosome segregation post-septationally by translocating trapped DNA across the membrane. Unlike E. coli , B. subtilis contains a second uncharacterized FtsK/SpoIIIE-like protein, SftA (formerly YtpS). We report that SftA plays a role similar to FtsK during each cell cycle but cannot substitute for SpoIIIE in rescuing trapped chromosomes. SftA colocalizes with FtsZ at nascent division sites but not with SpoIIIE at sites of chromosome trapping. SftA mutants divide over unsegregated chromosomes more frequently than wild-type unless recA is inactivated, suggesting that SftA, like FtsK, stimulates chromosome dimer resolution. Having two FtsK/SpoIIIE paralogues is not conserved among endospore-forming bacteria, but is highly conserved within several groups of soil- and plant-associated bacteria.     

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double‐stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.     

A defined terminal region of the E. coli chromosome shows late segregation and high FtsK activity

Background

The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV.

Methodology

We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a ∼400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPS-recognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions.

Significance

Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last steps of chromosome segregation and to their coupling with cell division by FtsK.     

Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV

The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.     

Asymmetric DNA requirements in Xer recombination activation by FtsK

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis.     

Identification of the FtsK sequence-recognition domain

FtsK is a prokaryotic multidomain DNA translocase that coordinates chromosome segregation and cell division. FtsK is membrane anchored at the division septum and, guided by highly skewed DNA sequences, translocates the chromosome to bring the terminus of replication to the septum. Here, we use in vitro single-molecule and ensemble methods to unveil a mechanism of action in which the translocation and sequence-recognition activities are performed by different domains in FtsK.     

FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.     

FtsK controls metastable recombination provoked by an extra Ter site in the Escherichia coli chromosome terminus

The FtsK protein is required for septum formation in Escherichia coli and as a DNA translocase for chromosome processing while the septum closes. Its domain of action on the chromosome overlaps the replication terminus region, which lies between replication pause sites TerA and TerC. An extra Ter site, PsrA*, has been inserted at a position common to the FtsK and terminus domains. It is well tolerated, although it compels replication forks travelling clockwise from oriC to stall and await arrival of counter-clockwise forks. Elevated recombination has been detected at the stalled fork. Analysis of PsrA*-induced homologous recombination by an excision test revealed unique features. (i) rates of excision near PsrA* may fluctuate widely from clone to clone, a phenomenon we term whimsicality, (ii) excision rates are nevertheless conserved for many generations, a phenomenon we term memorization; their metastability at the clone level is explainable by frequent shifting between three cellular states--high, medium and low probability of excision, (iii) PsrA*-induced excision is RecBC-independent and is strongly counteracted by FtsK, which in addition is involved in its whimsicality and (iv) whimsicality disappears as the distance from the pause site increases. Action of FtsK at a replication fork was unexpected because the factor was thought to act on the chromosome only at septation, i.e. after replication is completed. Idiosyncrasy of PsrA*-induced recombination is discussed with respect to possible intermingling of replication, repair and post-replication steps of bacterial chromosome processing during the cell cycle.     

FtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein

Escherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N-terminal transmembrane domain (FtsK(N)) is essential for septum formation, whereas its C-terminal domain (FtsK(C)) is required for chromosome dimer resolution by XerCD-dif site-specific recombination. FtsK(C) is an ATP-dependent DNA translocase. In vitro and in vivo data point to a dual role for this domain in chromosome dimer resolution (i) to directly activate recombination by XerCD-dif and (ii) to bring recombination sites together and/or to clear DNA from the closing septum. FtsK(N) and FtsK(C) are separated by a long linker region (FtsK(L)) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsK(L) and/or of FtsK(C), of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsK(C). Phenotypic characterization of the mutants indicated a role for FtsK(L) in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsK(C), mutants were found that can perform only one or the other of these two functions, which allowed their separation in vivo for the first time.     

Unlinking chromosome catenanes in vivo by site-specific recombination

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.