Frequent exchange of the DNA polymerase during bacterial chromosome replication

Abstract

The replisome is a multiprotein machine that carries out DNA replication. In Escherichia coli, a single pair of replisomes is responsible for duplicating the entire 4.6 Mbp circular chromosome. In vitro studies of reconstituted E. coli replisomes have attributed this remarkable processivity to the high stability of the replisome once assembled on DNA. By examining replisomes in live E. coli with fluorescence microscopy, we found that the Pol III* subassembly frequently disengages from the replisome during DNA synthesis and exchanges with free copies from solution. In contrast, the DnaB helicase associates stably with the replication fork, providing the molecular basis for how the E. coli replisome can maintain high processivity and yet possess the flexibility to bypass obstructions in template DNA. Our data challenges the widely-accepted semi-discontinuous model of chromosomal replication, instead supporting a fully discontinuous mechanism in which synthesis of both leading and lagging strands is frequently interrupted.DOI: http://dx.doi.org/10.7554/eLife.21763.001

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

“…Frequent polymerase exchange and uncoupled lagging‐strand synthesis have also been detected during E. coli replication. In contrast to the exchange of individual T7 polymerases, in E. coli , entire Pol III* (Pol III HE lacking β clamp) complexes exchange . Consistent with the variation seen in T7 polymerase number, more than one Pol III* subassembly has been detected at the replication fork .…”

“…The foundation of our understanding of replication mechanism derives from several seminal experiments conducted in the second half of the 20th century which suggested DNA replication is performed by a single static complex with highly defined operating principles. In contrast to this view, single‐molecule observations have revealed that dynamic exchange of core components, pausing events, and several types of DNA loops may underlie coordination of daughter‐strand synthesis . Individually, these events support different mechanistic models which cannot always be reconciled in a single reaction mechanism.…”

“…Nevertheless, this multi‐pathway view of replication is often at odds with experimental observations that still dominate our textbook view of the process. Given the numerous compelling studies supporting this dynamic view we must re‐examine the early experiments that underlie our core theories.…”

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

“…Such a deterministic model violates fundamental chemical principles (van Oijen and Dixon, 2015), not to mention the pragmatism of evolution, and it has been challenged by three recent single-molecule studies that uncover alternate pathways. The first two studies demonstrate facile exchange between complete replicase units at the fork and in solution, on the seconds timescale, both in vivo (Beattie et al 2017) and in vitro (Lewis et al 2017). The third study (Graham et al 2017) suggests that even under conditions with no free polymerases in solution, the DnaB helicase can transiently uncouple and proceed more slowly ahead of the replicase at the apex of the replication fork.…”

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

“…The bacterial replisome is the prototype of a fixed structural assembly of both leading and lagging strand polymerases with the clamploader. However, recent studies indicate that there is a frequent exchange of the bacterial polymerase during replication [175]. In the case of Pol ε, a similar situation could exist in terms of disengagement, such that uncoupling might be more frequent than expected even in the absence of replication stress.…”