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

Abstract

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...

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

“…Our data suggest that FtsK is preferentially loaded at KOPS only under conditions in which ATP hydrolysis is prevented. This finding contrasts with bulk biochemical assays, which have suggested that FtsK loads at KOPS when ATP is used as the nucleotide cofactor (9). One explanation for this discrepancy is that prior bulk assays used relatively short DNA substrates with a correspondingly high density of KOPS sites, ranging from 1 KOPS per 30 bp up to 1 KOPS per 336 bp.…”

“…Given the abundance of ATP in vivo, and the finding that KOPS recognition is suppressed even in ADP/ATP mixtures, we speculate that cooperative stepwise assembly of FtsK hexamers from monomeric subunits at KOPS may be used as a mechanism for regulating the initiation of translocation, as previously suggested (5,6,9,23). Such a model would presume that complete hexamer assembly from monomers is inefficient on nonspecific DNA and that ATP hydrolysis is inhibited until the hexamer is fully assembled at KOPS.…”

“…Early reports suggested that FtsK could recognize KOPS during translocation, enabling it to reorient while translocating (7,8,14,19). However, other studies have suggested that KOPS acts only as a loading site for FtsK (9,13).…”

“…In vitro work has focused on the C-terminal FtsKαβγ motor domain, referred to as FtsK 50C , or similar constructs (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). FtsK 50C is a DNA-dependent ATPase, which can activate XerCDdif recombination (6).…”

FtsK translocation permits discrimination between an endogenous and an imported Xer/ dif recombination complex

“…However, translocation appears to be a prerequisite for activation in the natural situation (i.e., when FtsKγ is linked to the FtsK motor). This can be inferred from the incapacity of FtsK mutants defective in translocation (i.e., unable to hydrolyze ATP) to induce recombination (29,30). We further show that an active FtsK motor also fails to activate recombination if unable to stop at the recombination complex.…”