Abstract
Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved. A site-specific recombination system catalyses this dimer-resolution reaction at the chromosomal site dif. In Escherichia coli, two tyrosine-family recombinases, XerC and XerD, bind to dif and carry out two pairs of sequential strand exchange reactions. However, what makes the reaction unique among site-specific recombination reactions is that the first step, XerD-mediated strand exchange, relies on interaction with the very C-terminus of the FtsK DNA translocase. FtsK is a powerful molecular motor that functions in cell division, co-ordinating division with clearing chromosomal DNA from the site of septation and also acts to position the dif sites for recombination. This is a model system for unlinking, separating and segregating large DNA molecules. Here we describe the molecular detail of the interaction between XerD and FtsK that leads to activation of recombination as deduced from a co-crystal structure, biochemical and in vivo experiments. FtsKγ interacts with the C-terminal domain of XerD, above a cleft where XerC is thought to bind. We present a model for activation of recombination based on structural data.
Highlights
The majority of bacterial species have circular DNA genomes
The activation of recombination at dif requires the interaction of XerD with the γdomain of FtsK. To understand how this interaction can lead to activation of recombination, a fusion protein between the C-terminal domain of XerD, which contains the catalytic residues required for DNA cleavage and strand exchange, and FtsKγwas produced (XerDC–γ) and crystallized
The structure of the XerDCFtsKγfusion protein was solved by molecular replacement and refined to a resolution of 2.3 Å (Fig. 1), using the previously reported XerD structure (1A0P) and the NMR structure of the E. coli FtsKγdomain (2VE8) as search
Summary
The majority of bacterial species have circular DNA genomes. Prior to cell division each circular chromosome must be entirely replicated, unlinked and segregated to ensure that each daughter cell inherits a full genome complement. During or following replication DNA repair processes involving homologous recombination can produce a chromosomal crossover, and any odd number of these events between the circular DNA molecules results in a chromosome dimer[1] Evidence suggests that this occurs with a probability of 17–40% for each cell cycle[2,3]. Most bacteria encode two tyrosine family recombinases XerC and XerD which bind to the specific chromosomal site dif located in the replication terminus region[5,6], there are notable exceptions where a single Xer protein carries out the reaction[7,8,9]. The C-terminal domain of FtsK forms a DNA translocase motor, which hexamerises, and was found to be essential for chromosome dimer resolution through activation of the XerD recombinase[25,26,27]. Three FtsKγdomains bind to each KOPS site, which leads to loading and hexamerisation of the FtsK motor domains to one side of the KOPS sequence so that subsequent translocation is always directed towards the dif site[31]
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