1. Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier.
- Author
-
Berghuis BA, Dulin D, Xu ZQ, van Laar T, Cross B, Janissen R, Jergic S, Dixon NE, Depken M, and Dekker NH
- Subjects
- Base Sequence, Binding Sites, Chromosomes, Bacterial chemistry, Chromosomes, Bacterial metabolism, DNA, Bacterial chemistry, DNA, Circular chemistry, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Escherichia coli genetics, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, DNA Replication, DNA, Bacterial metabolism, DNA, Circular metabolism, DNA-Binding Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism
- 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.
- Published
- 2015
- Full Text
- View/download PDF