Two mechanisms coordinate replication termination by theEscherichia coliTus–Tercomplex

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.

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

“…Coordination of leading-and lagging-strand synthesis is critical for high processivity of the replisome (5,(31)(32)(33)(34)(35)(36). However, the enzymatic events on the lagging and leading strands are not equivalent.…”

What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments

“…Encounter of Tus-Ter complex by the heterologous bacteriophage T7 replisome Pandey et al (2015) use a single-molecule flow-stretching assay (Figure 3(a)) as well as a quenched flow assay (Figure 3(b)) to examine the efficiency and details of Tus-Ter blocking the progression of the well-characterized heterologous bacteriophage T7 helicase and DNA polymerase. The use of a heterologous system is warranted because it is possible to synchronize initiation of DNA replication in the T7 system by preassembly of the replisome without Mg 2þ , in contrast to in vitro replication by the E. coli replisome where loading of the DnaB helicase is inefficient in the absence of a natural origin of replication.…”

“…Here, we specifically discuss three recently published studies designed to further examine or integrate these models and unravel the mechanism of polar Tus-Ter replication fork arrest. A first study, by Pandey et al (2015), examines the polar interaction of Tus-Ter when the heterologous bacteriophage T7 replisome, the isolated T7 replicative helicase, or the T7 DNA polymerase collide with this DNA roadblock. Two approaches are used here: a single-molecule flow stretching assay (Figure 3(a)), as well as a quench flow bulk-phase assay with single base pair resolution (Figure 3(b)).…”

“…Two approaches are used here: a single-molecule flow stretching assay (Figure 3(a)), as well as a quench flow bulk-phase assay with single base pair resolution (Figure 3(b)). In a second study, Berghuis et al (2015) investigate the sequence of events that lead to polar blocking of strand separation by probing the Tus-Ter Elshenawy et al (2015) and Pandey et al (2015). A forked double-stranded (ds) DNA template is anchored to a micron-sized bead and the surface of a flow-cell.…”

“…(b) The quench flow assay as performed by Pandey et al (2015), where the T7 replication machinery (helicase, polymerase or both) initiates synchronously on short $60 bp forked dsDNA substrates containing a single Ter site. The reaction is stopped at discrete time intervals and products are gel-imaged.…”