FtsK translocation on DNA stops at XerCD-dif - PubMed (original) (raw)
FtsK translocation on DNA stops at XerCD-dif
James E Graham et al. Nucleic Acids Res. 2010 Jan.
Abstract
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 FtsKgamma regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.
Figures
Figure 1.
Stoppage of FtsK translocation by the XerCD-dif. (a) Left panel: Translocation was measured by displacement of a radio labelled TFO in the presence or absence of bound recombinases (‘Materials and Methods’ section). Substrates contained a triplex binding site biased towards one end of a linear DNA. The hexameric FtsK ring is represented as a cone with only two γ-subdomains shown for clarity. The inset shows the sequence of the dif site in ‘XerC-first’ orientation, XerC being the recombinase that would first be encountered by FtsK. Positions of the scissile phosphates in the recombination reaction are indicated with black triangles, and the central region with a grey box. We define the ‘inner’ face of XerC/D as that which faces the central region. (a) Right panel: Model of the hexameric FtsK motor loaded on KOPS DNA. This model is a composite of the crystal structures of the hexameric αβ-subdomains (30) and the trimeric γ-subdomains bound to KOPS (21) with B-DNA modelled through the DNA channel. The model represents an ‘initiation complex’ for FtsK translocation. The ‘handle’ of FtsK is also indicated. (b) The stoppage by XerCD was measured on substrates carrying the dif site in an ‘XerD-first’ or ‘XerC-first’ orientation (cartoons, left). Representative gels are shown. Time-courses (0, 1 and 2 min), with raw displacement values (with unbound TFO at zero time subtracted) from a single experiment, are shown beneath each gel. Bars show normalized stoppage values from three experiments expressed as a percentage of a control experiment lacking recombinase at 1 and 2 min (±SD) (‘Materials and Methods’ section). (c) MatP–matS complex does not stop FtsK translocation. Normalized stoppage values are shown, as per (b). (d) EcoKI displaces XerCD from dif. Translocation by EcoKI was investigated on the substrate containing dif in the ‘XerD-first’ orientation, with and without bound XerCD. Translocation initiates from the EcoKI site bound by the methyltransferase (rectangular box and ‘M’), and the cartoon shows the looping of DNA by the translocating HsdR subunits (R). Triplex displacement profiles are shown for 10-min reactions performed in triplicate.
Figure 2.
Stoppage of translocation by XerC and XerD recombinases bound alone to their cognate dif half sites. Triplex displacement by FtsK was measured on substrates bearing a complete dif site but with only one of the Xer recombinases bound (i–iv), or on half dif sites containing only 2 bp of the central region (v–viii). The composition and orientation of these sites is shown schematically. Normalized displacement values are expressed as per Figure 1.
Figure 3.
Specific FtsKγ-XerD interaction is not required for FtsK translocation stoppage. Triplex displacement by FtsK and FtsKΔγ was measured on a substrate with dif in either orientation (cartoons), and in the presence of XerC and/or XerD recombinases, or a quadruple mutant of XerD (R288K Q289R Q292E Q293R), XerDQ.
Figure 4.
Translocation stoppage at Cre recombinase bound to loxP. The ability of FtsK to displace Cre from loxP was measured on substrates with loxP in either orientation. The orientation of loxP is defined by the asymmetry of the nucleotide sequence close to the scissile phosphate, which results in preferential cleavage of one scissile phosphate (black arrow; ref. 32). We denote the central region and define the inner and outer faces of Cre in the same manner as in Figure 1a. The Cre monomer that mediates the first cleavage is indicated by a darker shade of grey. ‘P-first’ and ‘NP-first’ denote the orientations of loxP in which FtsK approaches preferentially and non-preferentially cleaved half sites of the loxP first, respectively. We also tested displacement on a substrate carrying a half-loxP site that contains the preferentially cleaved scissile phosphate. This half-site was also tested in both orientations, so that FtsK approached the Cre monomer either on its inner or outer face.
Figure 5.
Recombinases deficient in DNA synapsis or cleavage stop translocation of FtsK. (a) Triplex displacement was measured on a substrate bearing a loxP site with the active Cre monomer first (orientation ‘P’, cartoon), in the presence of either wild-type Cre or a synapsis-defective Cre mutant (A36V). (b) Normalized triplex displacement by FtsK on a dif substrate in the presence of combinations of wild-type XerC, XerD and catalytic mutants XerCK172Q and XerDK172Q.
Figure 6.
FtsK ATPase activity is reduced upon stoppage by XerCD-dif. The ATPase activity of FtsK was measured on short linear substrates bearing a KOPS loading site with a 25-bp loading arm, and dif site positioned 5 bp away from the KOPS (cartoons) using an NADH coupled assay. Loading on KOPS directs FtsK translocation towards dif, as indicated by the arrow. The experiments were performed with substrates bearing a full dif site in XerD-first (a) or XerC-first (b) orientation and on an XerD dif half-site in both orientations (c) (d) and substrates lacking dif and carrying either a 39 bp (e) or 5 bp (f) extension downstream of KOPS. The traces begin ∼30 s after the addition of 2 mM ATP. ATPase rates (µM/s) are shown in boxes next to each graph; they were determined from linear fits to the average of three traces; SDs were <0.05 (µM/s). (g) ATPase activity of FtsK variants on a short substrate carrying dif in XerD-first orientation [as in (a)].
References
- Bigot S, Sivanathan V, Possoz C, Barre FX, Cornet F. FtsK, a literate chromosome segregation machine. Mol. Microbiol. 2007;64:1434–1441. - PubMed
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