Continuous Assays for DNA Translocation Using Fluorescent Triplex Dissociation: Application to Type I Restriction Endonucleases

McClelland, Sarah E.; Dryden, David T. F.; Szczelkun, Mark D.

doi:10.1016/j.jmb.2005.03.018

Cited by 55 publications

(111 citation statements)

References 73 publications

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“…At ≤50 nM FtsK monomer, triplex displacement was incomplete: a fast exponential phase preceded a >100-fold slower linear phase. These profiles are characteristic of stepwise translocation initiating from a specific point relative to the triplex (13)(14)(15). At 100 nM FtsK the amplitude of the first phase increased moderately but the second phase increased in both rate and amplitude to give >80% displacement after ∼3 min.…”

Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 96%

“…Rebinding of the TFO does not occur (13). If FtsK initiates from a specific location relative to the TBS, a characteristic lag in the displacement kinetics can be used to compute the translocation velocity (14). We performed a series of experiments on linear DNAs containing a TBS at fixed distances from three overlapping KOPS ("triple KOPS," or tKOPS, GGGCAGGGCAGGGCAGGG).…”

Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 99%

“…We were concerned that at higher FtsK concentrations the profiles did not conform to models for a linear translocase initiating from a single site (14). Although none of our DNA substrates contained additional "bona fide" KOPS, we reasoned that the affinity for nonspecific DNA-perhaps sequences that resemble KOPS (SI Text)-is similar: high FtsK concentration would lead to assembly of hexamers elsewhere, resulting in distributive displacement kinetics.…”

Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 99%

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Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

Graham

Sherratt

Szczelkun

2010

Proc. Natl. Acad. Sci. U.S.A.

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FtsK is a homohexameric, RecA-like dsDNA translocase that plays a key role in bacterial chromosome segregation. The FtsK regulatory γ-subdomain determines directionality of translocation through its interaction with specific 8 base pair chromosomal sequences [(KOPS); FtsK Orienting / Polarizing Sequence(s)] that are cooriented with the direction of replication in the chromosome. We use millisecond-resolution ensemble translocation and ATPase assays to analyze the assembly, initiation, and translocation of FtsK. We show that KOPS are used to initiate new translocation events rather than reorient existing ones. By determining kinetic parameters, we show sigmoidal dependences of translocation and ATPase rates on ATP concentration that indicate sequential cooperative coupling of ATP hydrolysis to DNA motion. We also estimate the ATP coupling efficiency of translocation to be 1.63-2.11 bp of dsDNA translocated/ATP hydrolyzed. The data were used to derive a model for the assembly, initiation, and translocation of FtsK hexamers.F tsK is a highly conserved dsDNA translocase involved in chromosome unlinking in bacteria, coordinating the final stages of chromosome segregation with cytokinesis, and performing chromosome dimer resolution by interacting with XerCD (1). Escherichia coli FtsK is a 1,369 amino-acid protein that comprises an N-terminal integral membrane domain that localizes FtsK to the septum early in cell division; a long linker of unknown function; and a C-terminal translocase domain (FtsK C ) that forms hexameric rings on dsDNA. FtsK C has been shown in singlemolecule assays to translocate on dsDNA at speeds of ∼5 kb s −1 and against a stall force of ∼60 pN (2-7). FtsK C comprises FtsKαβ, which constitute the hexameric motor; and FtsKγ, a winged-helix domain that both interacts with XerCD bound to dif to promote chromosome unlinking and recognizes a specific 8 bp sequence, KOPS (FtsK Orienting/Polarizing Sequence; 5′-GGG[C/A]AGGG-3′). FtsKγ binds to KOPS as a trimer, with three FtsKγ winged-helix modules interacting with consecutive GGG, MA, and GGG elements (8). KOPS were identified by bioinformatic (7) and genetic (3) screens as sequences polarized towards dif. Thus, KOPS orient FtsK towards XerCD-dif.FtsK 50C , a biochemically active form of FtsK C , has been used for most biochemical and single-molecule studies; however, the protein is subject to aggregation and large particles are visible under the light microscope (5-7). Initial reports suggested that FtsK 50C reverses direction when KOPS are encountered in "nonpermissive" orientation during translocation (5, 7, 9). Conversely, structural and biochemical studies showed that KOPS are loading sites that establish oriented translocation (2, 8). Reversal of translocation at nonpermissive KOPS has been explained by the translocating unit being a double-hexamer, with a switch in the activity between motors (7, 10), or by assuming that a translocating hexamer encounters a second hexamer bound to, or translocating from, a nonpermissive KOPS (8). Biochemical ex...

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Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 96%

Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 99%

Section: Kops-dependent and -Independent Initiation Of Translocation mentioning

confidence: 99%

See 1 more Smart Citation

Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

Graham

Sherratt

Szczelkun

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Fluorescent triplex substrates were designed as described in Levy et al 19 and prepared following the method developed by McClelland et al 44 . Displacement reactions were conducted at 25 °C in 50 mM Tris (pH 7.5), 5 mM MgCl 2 , 3 mM ATP, 0.1 mg ml −1 BSA, 10 nM 5′-6-rhodamine triplex substrate and 20-50 nM SpoIIIE or 100nM SpoIIIE-SK chimera.…”

Section: Triplex Displacement Assaysmentioning

confidence: 99%

Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

Ptacin

Nollmann

Becker

et al. 2008

Nat Struct Mol Biol

165

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In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to study the properties of DNA translocation by SpoIIIE in vitro and in vivo. These data allow us to propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.The segregation and exchange of genetic material are central to the processes of cell division and evolution. Although mechanisms of genetic transfer between cellular compartments are diverse, all require the movement of DNA across cellular membranes. A dramatic example of intercellular transmembrane DNA transport is the segregation of chromosomes during sporulation in Bacillus subtilis. During this process, newly replicated chromosomes are rearranged into an elongated structure, or axial filament, in which the origin of replication of each chromosome is tethered to opposite cell poles 1,2 . Next, the division plane is relocated to one pole, creating two asymmetric cellular compartments (the forespore and the mother cell) and trapping the origin-proximal 30% of one chromosome within the forespore 3,4 . The septally

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“…As the enzyme tracks the DNA helix, supercoiling can build up in the extruded loops. How the enzyme can translocate for up to 50,000 base pairs (bp) (García and Molineux 1999) and at speeds of up to 1000 bp per second (Seidel et al 2004) in the face of the torsional stress induced by this supercoiling is not known, although it seems that the enzyme makes many abortive attempts to initiate the translocation (McClelland et al 2005).…”

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confidence: 99%

Structure and operation of the DNA-translocating type I DNA restriction enzymes

Kennaway¹,

Taylor²,

Song³

et al. 2012

Genes Dev.

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Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

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Continuous Assays for DNA Translocation Using Fluorescent Triplex Dissociation: Application to Type I Restriction Endonucleases

Cited by 55 publications

References 73 publications

Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

Structure and operation of the DNA-translocating type I DNA restriction enzymes

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