Asymmetric DNA requirements in Xer recombination activation by FtsK
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.
Full-textDOI: · Available from: François-Xavier Barre, May 29, 2015
SourceAvailable from: François CornetBiophysical Journal 01/2013; 104(2):76-. DOI:10.1016/j.bpj.2012.11.455 · 3.83 Impact Factor
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ABSTRACT: A global view of bacterial chromosome choreography during the cell cycle is emerging, highlighting as a next challenge the description of the molecular mechanisms and factors involved. Here, we review one such factor, the FtsK family of DNA translocases. FtsK is a powerful and fast translocase that reads chromosome polarity. It couples segregation of the chromosome with cell division and controls the last steps of segregation in time and space. The second model protein of the family SpoIIIE acts in the transfer of the Bacillus subtilis chromosome during sporulation. This review focuses on the molecular mechanisms used by FtsK and SpoIIIE to segregate chromosomes with emphasis on the latest advances and open questions. © 2015 S. Karger AG, Basel.
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ABSTRACT: Circular chromosomes can form dimers during replication and failure to resolve those into monomers prevents chromosome segregation, which leads to cell death. Dimer resolution is catalysed by a highly conserved site-specific recombination system, called XerCD-dif in Escherichia coli. Recombination is activated by the DNA translocase FtsK, which is associated with the division septum, and is thought to contribute to the assembly of the XerCD-dif synapse. In our study, direct observation of the assembly of the XerCD-dif synapse, which had previously eluded other methods, was made possible by the use of Tethered Particle Motion, a single molecule approach. We show that XerC, XerD and two dif sites suffice for the assembly of XerCD-dif synapses in absence of FtsK, but lead to inactive XerCD-dif synapses. We also show that the presence of the γ domain of FtsK increases the rate of synapse formation and convert them into active synapses where recombination occurs. Our results represent the first direct observation of the formation of the XerCD-dif recombination synapse and its activation by FtsK.Nucleic Acids Research 11/2013; 42(3). DOI:10.1093/nar/gkt1024 · 8.81 Impact Factor