| Autor: |
Berghuis BA; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Dulin D; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Xu ZQ; Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia., van Laar T; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Cross B; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Janissen R; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Jergic S; Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia., Dixon NE; Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, Australia., Depken M; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands., Dekker NH; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands. |
| Abstrakt: |
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. |
