Abstract
The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus–Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus–Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus–Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
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Acknowledgements
We thank J. Cnossen for his countless efforts in adjusting the MT software to our needs, J. Kerssemakers for discussions and S. Hamdan for his contribution to the discussion by generously sharing his own experimental observations. This study was supported by a grant from the Australian Research Council (DP0877658) to N.E.D. and by Vici and TOP grants from the Netherlands Organisation for Scientific Research (NWO) and ERC Starting Grant DynGenome from the European Research Council to N.H.D.
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N.E.D. and N.H.D. designed the research. B.A.B. and N.H.D. designed the experiments. D.D. designed and assembled the magnetic tweezers apparatus. B.A.B. performed the experiments. B.A.B., B.C., T.v.L., R.J. and N.H.D. designed, and B.C. and T.v.L. made, the DNA hairpin constructs. Z.-Q.X. and S.J. purified the Tus proteins. B.A.B. and M.D. analyzed the data. M.D. developed the application of MLE to force spectroscopy data. B.A.B., D.D., M.D. and N.H.D. interpreted the data. S.J. and N.E.D. contributed to discussions concerning the model and in vivo observations. B.A.B., N.E.D. and N.H.D. wrote the paper.
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Berghuis, B., Dulin, D., Xu, ZQ. et al. Strand separation establishes a sustained lock at the Tus–Ter replication fork barrier. Nat Chem Biol 11, 579–585 (2015). https://doi.org/10.1038/nchembio.1857
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DOI: https://doi.org/10.1038/nchembio.1857
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