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Nucleic Acids Res. 2015 Jul 13;43(12):5924-35. doi: 10.1093/nar/gkv527. Epub 2015 May 24.

Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

Author information

1
Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA pandeyma@rwjms.rutgers.edu.
2
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
3
Centre for Medical and Molecular Bioscience, University of Wollongong, New South Wales 2522, Australia.
4
Centre for Medical and Molecular Bioscience, University of Wollongong, New South Wales 2522, Australia nickd@uow.edu.au.
5
Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA patelss@rutgers.edu.

Abstract

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus-Ter complex. The T7 replisome was arrested at the non-permissive end of Tus-Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus-Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

PMID:
26007657
PMCID:
PMC4499146
DOI:
10.1093/nar/gkv527
[Indexed for MEDLINE]
Free PMC Article

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