Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.
Bottom Line: 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.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.
Affiliation: Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA firstname.lastname@example.org.Show MeSH
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Mentions: We next used a rapid quenched flow assay to follow progressive DNA synthesis at single base resolution, allowing us to study the encounter of the T7 helicase–polymerase with Tus–TerB at high spatial and temporal resolution. The 22-bp TerB sequence was introduced in either the permissive or non-permissive orientation at a specific position in the middle of a 60-bp dsDNA region of the synthetic replication fork substrate (Figure 2 and Supplementary Table S1). The replication fork contained a 35-nt 5′-tail for loading of the helicase and a 24-bp primer/template for loading the polymerase (Figure 3A, B). The replication fork was pre-incubated with Tus (380 nM) and the T7 DNA polymerase and helicase in the presence of dTTP, but without Mg2+. These conditions promote preassembly of the replication proteins on the DNA and allow synchronization of the DNA unwinding/synthesis reactions initiated with Mg2+ and dNTPs. The components were mixed in a chemical quenched-flow instrument and quenched after 0.004 to 600 s before analysis of primer extension at single base resolution on a DNA sequencing gel (Figure 3C).
Affiliation: Department of Biochemistry and Molecular Biology, Rutgers, the State University of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA email@example.com.