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Mentions: The effect of top and bottom-strand gaps on recombination was attenuated when they were displaced away from dif (Figures 5 and 7), indicating that they locally influence the recombination process. Activation of recombination by FtsK requires a direct contact between FtsKγ and XerD (17,18). This contact has to occur in cis, since FtsK cannot activate recombination when loaded on the XerC side of the complex on short asymmetric substrates, whether KOPS are present (data not shown) or not (25). FtsKγ is connected to the FtsK50C engine by a region of approximately 15 aa (including a large proportion of glycine residues), which is probably unstructured and could extend to 4 or 5 nm. This should be sufficient to allow FtsKγ to contact XerD in cis, even if the FtsK motor stalled 3 bp before the (XerCD–dif)2 complex, as would be the case for 2 nt gaps positioned at 0 or 1 bp from dif (Figure 8). Thus, it is unlikely that 2 nt gaps in the immediate vicinity of dif impede the interaction of FtsKγ and XerD. It would be therefore tempting to propose that FtsK50C needs to change the conformation of the (XerCD–dif)2 complex in addition to contacting XerD. The top and bottom-strands asymmetry could then be explained by the fact that some mechanical stress needs to be propagated through the bottom strand of the substrate, i.e. the strand cleaved by XerD, for this change of conformation to occur. Indeed, the minimum size of bottom-strand gaps abolishing Xer recombination is 2 nt (Figure 5), which fits remarkably well with the step size of FtsK monomers, as estimated from crystallographic data (19). Two types of mechanical stress could occur, torsion or flexion. Torsion is ruled out because (i) the motion of FtsK50C induces very little rotation relative to the DNA (21) and (ii) the presence of nicks has no effect on the efficiency of recombination (Figure 5). Flexion is also very unlikely since FtsK can translocate on single-stranded DNA in both the 5′–3′ and 3′–5′ orientation along at least 5 nt, which should allow it to push on the (XerCD–dif)2 synapse, even if a few nucleotides are missing in the vicinity of dif (Figure 7). In addition, we observed that XerC and XerD can stop the translocation of FtsK on DNA when bound to dif (Bonné,L. et al., unpublished data), which should limit the amount of torsion and flexion that FtsK could introduce in the vicinity of the complex. Therefore, we propose that the specific nucleotides we identified in the immediate vicinity of dif (Figure 8) serve to stabilize FstK at the proximity of the (XerCD–dif)2 complex, to allow for a sufficiently long and stable contact between FtsKγ and XerD for recombination to occur.
Asymmetric DNA requirements in Xer recombination activation by FtsK
Bottom Line: 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.
Affiliation: CNRS, Centre de Génétique Moléculaire, FRE 3144, 91198 Gif-sur-Yvette, France.
Abstract: 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.
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