Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase - PubMed (original) (raw)

Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase

James Yates et al. Mol Microbiol. 2006 Mar.

Abstract

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.

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Figures

Fig. 1

Fig. 1

FtsK50C stimulates XerD-mediated cleavage of BSN.

  1. Schematic of XerCD-mediated cleavages of nicked suicide substrates, BSN and TSN. XerC (circle) binds to the left half-site of dif while XerD (square) binds to the right half-site. A 6 bp central region separates the two binding sites. The 28 bp dif site is flanked by 17 bp or 13 bp DNA segments. XerC and XerD cleave the top (grey) and bottom (black) strand of dif respectively. The position of the 5′ radiolabel is indicated by a star. The BSNe substrate carries a 205 bp extension adjacent to XerD binding site (not shown). Cleavage by either recombinase gives a diagnostic labelled DNA fragment with covalently attached recombinase.
  2. The 60 min 37°C reactions of the indicated reagents were analysed on a 0.1% SDS-6% PAGE. The levels of FtsK50C-mediated stimulation of DNA cleavage by XerD and XerC are shown, as are the ratios of cleavage by XerD as compared with XerC. The levels of FtsK50C-mediated stimulation of DNA cleavage by XerD varied between 2.5- and 32-fold after 60 min reactions in different experiments. In part, this results from day-to-day variations in FtsK50C-specific activity. Within experiments using a given set of protein dilutions, cleavage activities can be reliably compared.
  3. Time-course of XerD-mediated cleavage of BSN substrate in the presence and absence of FtsK50C. The percentage of substrate DNA converted to BSN-XerD with respect to time is plotted. The level of stimulation by FtsK50C as judged by initial rates is > 10-fold in this experiment.
  4. XerCD cleavage reactions with TSN substrate.

Fig. 2

Fig. 2

A 62-amino-acid region of the FtsKCγ subdomain can stimulate DNA cleavage by XerD.

  1. A schematic of FtsK, FtsK50C and the maltose-binding protein (MBP) fusion derivatives.
  2. The 60 min 37°C reactions of BSN with the indicated reagents were analysed by SDS-PAGE as in Fig. 1.

Fig. 3

Fig. 3

The FtsKCγ subdomain can stimulate intermolecular recombination between BSN and intact _dif_-DNA substrates.

  1. The 60 min 37°C reactions containing radiolabelled BSN and 197_dif_200, an unlabelled intact linear DNA duplex, and the indicated reagents were analysed by 0.1% SDS-4% PAGE. BSN-C, covalent complex of BSN fragment and XerC; BSN-D, covalent product of BSN and XerD; LP, nicked or gapped recombinant product of BSN and 197_dif_200; LP-D, XerD covalent complex of gapped LP; HJ, nicked or gapped HJ; HJ-C, covalent complex of XerC with a HJ that has lost a DNA arm; HJ-D, covalent complex of XerD and a gapped HJ (see C).
  2. Time-course of reaction of radiolabelled BSN, 197_dif_200 DNA, and XerCD in the absence (left) and presence of FtsK50C (right). DNA species as labelled in (A).
  3. The proposed sequence of steps leading to generation of the observed radiolabelled products initiated by XerD. Completion of a pair of strand exchanges by XerD requires that the 3 nt fragment (represented by three dots) released by BSN cleavage by XerD does not diffuse out of the synaptic complex so that its 3′ end can be used to attack the phosphotyrosyl bond on 197_dif_200. A gapped HJ can arise by hydrolysis of the phosphotyrosine in HJ-D. The nick in BSN and subsequent products is shown by a small arrow.

Fig. 4

Fig. 4

Synapsis-dependent and -independent reactions. Varying concentration of TSN (top) or BSN (bottom), radiolabelled with an invariant concentration of radiolabelled DNA. Reactions were analysed by SDS-PAGE after 3 min, 15 min and 60 min reactions, with essentially the same overall results, although some of the 3 min reactions had very low levels of cleavage for one or the other recombinases. The 15 min (TSN) and 60 min (BSN) reactions are shown. The concentration dependence of the FtsKC-stimulated cleavage of BSN by XerD was essentially identical to BSN-D cleavage alone (the difference between the two BSN-D curves). FtsK50C had no effect on the TSN reactions or on cleavage of BSN by XerC (not shown).

Fig. 5

Fig. 5

The FtsKCγ subdomain interacts directly with XerD.

  1. XerCD interact in the absence of _dif_-DNA. Analysis by SDS-PAGE of recombinase interactions. 1. The indicated His-tagged recombinase is applied to cobalt agarose resin. L, protein loaded; F, flow through fraction; W, wash fraction. 2. Then the second indicated recombinase is applied to the washed column indicated in 1. E, proteins eluted by 300 mM imidazole; other abbreviations as in 1. Untagged XerCD did not bind to the columns under these binding conditions (controls).
  2. FtsK50C interacts with XerD. The sequential loading of His-tagged recombinase or FtsK50C to cobalt agarose resin, column washing, and then application of a second non-His-tagged protein, followed by 300 mM imidazole elution was as in (A). I–IV show the indicated protein combinations.
  3. MBPγ2 interacts with XerD. Sequential loading, washing and elution of the indicated proteins were as in (A). The elutions only are shown.
  4. MBPγ specifically competes with FtsK50C. Sequential loadings and elutions as indicated.

Fig. 6

Fig. 6

The C-terminal region of XerD interacts with FtsK50C.

  1. The indicated XerCD chimeras were tested for their ability to interact with immobilized HisFtsK50C by using the sequential loading and elution protocol described in Fig. 5. Abbreviations as in Fig. 5A. Per cent protein bound was calculated from percentage E/L.
  2. The indicated XerD variants carrying deletions at the C-terminus were tested for their ability to bind to immobilized HisFtsK50C. Abbreviations as in Fig. 5A.

Fig. 7

Fig. 7

XerD variants carrying substitutions at R288, Q289, Q292 and Q293 lack the ability to interact with FtsK50C.

  1. Amino acid sequence alignment of C-terminal regions of Ec XerD, Hi XerD and Ec XerC. The amino acid residue 282–293 region of Ec XerD (horizontal line) has been shown by deletion analysis (Fig. 6) to be necessary for the interaction with FtsK50C. Differences in amino acid sequence between Ec XerD and Hi XerD within this region are indicated (stars), as are the four residues targeted for mutagenesis (bold). The active site tyrosine (residue 279) is also indicated (ovoid).
  2. Interactions between the XerD variants and FtsK50C. The indicated XerD variants were assayed for their ability to bind to immobilized HisFtsK50C (open bars) and to stimulate cleavage of BSN in the presence of FtsK50C (grey bars). The activity of the variants is expressed as a percentage of the activity of wild-type Ec XerD.
  3. In vivo analysis of XerCD recombination at plasmid-borne dif in the presence of XerD or its variants. The SpR reporter plasmids carried either _dif_-KmR-dif (d) or _psi_-KmR-psi (p) cassettes in which two dif or two psi sites were directly oriented. Plasmid DNA preparations obtained from stationary-phase cultures of strains carrying reporter plasmids and vectors expressing XerD or XerD variants were analysed by agarose electrophoresis. The efficiencies of dif and psi plasmid resolution in the presence of XerD and XerD[KR..ER] were quantified by scoring for the loss of KmR marker upon transformation of the DNA samples shown into a Xer– strain. Five hundred transformants from each DNA sample were analysed.
  4. Efficiency of chromosome dimer resolution by FtsK-dependent recombination at dif, as judged by growth competition (Bigot et al., 2004). Competition was assessed at 10 generation intervals between the strains (KmR or TpR) expressing the indicated XerD variant and wild-type XerD from appropriate plasmids in a chromosomal XerD– background. A complementary competition experiment in which the chromosomal antibiotic resistance markers were exchanged led to the same results (not shown).

Fig. 8

Fig. 8

Mapping the FtsK γ region that interacts with XerD.

  1. Species specificity of the FtsKC–XerCD interaction between E. coli and H. influenzae in vitro. The indicated 60 min reactions were analysed by 0.1% SDS-6% PAGE.
  2. Amino acid sequence comparison between the γ2 regions of E. coli and H. influenzae. The elements of the Ec FtsKγ secondary structure derived from structure predictions are shown above the sequence; α-helices as horizontal grey lines, β-strand as a black line. The region of most notable sequence divergence is shown in bold font.
  3. FtsKγ variants were tested for binding to immobilized Ec HisXerD (open bars) and for their ability to stimulate _dif_-BSN DNA cleavage mediated by XerD (grey bars). The results of both assays are expressed as a percentage of the Ec MBPγ activity (100%).
  4. Paradigm of Cre–loxP recombination as applied to the activation of XerD by FtsKC. In a tetrameric recombination complex, only two of the four recombinase molecules are in an active state at any given time (either XerC or XerD in the case of XerCD). In the absence of FtsKC, XerCD–dif forms an ‘XerC-active’ synaptic complex preferentially (Hallet et al., 1999). In this conformation, XerC is active as a consequence of its C-terminal donor region interacting within the acceptor region of an XerD molecule bound to the same duplex; this positions the catalytic tyrosine of the active XerC molecule adjacent to the scissile phosphate (view from the C-terminal side of the complex). The switch from the ‘XerC-active’ to the ‘XerD-active’ configuration in a heterotetramer requires either dissolution of all recombinase–recombinase interactions and reformation of new interactions that lead to an altered DNA conformation, or dissociation of each DNA duplex from one of its recombinase binding sites and reformation of a complex with altered DNA paths. The action of FtsKC could be through pathway a, in which it directs the formation of the XerD-active state on duplex, and/or through pathway b, where the heterotetramer is the substrate for FtsKC action.

References

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