A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed

doi:10.1016/j.sbi.2013.06.018

Current Opinion in Structural Biology

Volume 23, Issue 5, October 2013, Pages 788-793

https://doi.org/10.1016/j.sbi.2013.06.018 Get rights and content

Highlights

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Single-molecule studies reveal dynamic behavior of the DNA-replication machinery.
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DNA polymerases are rapidly exchanged between the replication complex and solution.
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Multiple contact points allow both stable binding and rapid exchange of subunits.
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Dynamic exchange could be a general mechanism within multi-protein complexes.

Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multi-protein complexes that support multiple enzymatic activities. In this review, we will discuss single-molecule studies of the replisome, the protein complex responsible for the coordinated replication of double-stranded DNA. In particular, we will focus on new insights obtained into the dynamic nature of the composition of the DNA-replication machinery and how the dynamic replacement of components plays a role in the regulation of the DNA-replication process.

Section snippets

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

AMvO would like to acknowledge funding from the Netherlands Organization for Scientific Research (NWO; Vici 680-47-607) and the European Research Council (ERC Starting 281098).

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Cited by (22)

Bacterial chromosomes and their replication
2023, Molecular Medical Microbiology, Third Edition
This chapter reviews the current knowledge on the organization and replication of bacterial genomes with a special emphasis on the initiation of DNA replication. Most bacteria contain one chromosome, however a few species, like the human pathogen Vibrio cholerae, carry a secondary chromosome. We discuss structures of origins of replication, the function of initiator proteins, the cell cycle regulation of initiation of replication, and the loading and assembly of replisomes. This is predominantly described for Escherichia coli, but parallels are drawn to Bacillus subtilis along with important pathogenic bacteria when relevant.
Comparison of Bacterial and Eukaryotic Replisome Components
2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
Replisomes that duplicate genome DNA are composed of many different proteins that work together like gears in a machine. Sequence and structure comparisons reveal that replisome machines have evolved twice, once for bacteria and again for eukaryotes/archaea. This review compares the current state of knowledge on the structure and mechanisms of the central proteins that act directly at replication forks in bacteria to those of eukaryotes. The way these proteins organize their functions within a dynamic replisome machine is then compared for these two distinct domains of life.
A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome
2020, Molecular Cell
Citation Excerpt :
Instead, frequent turnover of the Pol III∗ in the replisome has been observed (Yuan et al., 2016; Beattie et al., 2017; Lewis et al., 2017), suggesting that the helicase acts as the central organizing structure as opposed to the polymerase. An explanation for this surprising level of plasticity can be found in the network of weak interactions that enable polymerases from solution to eventually replace those at the fork (Geertsema and van Oijen, 2013; Lewis et al., 2017). However, this explanation seems at odds with the putatively strong and stable interaction between multimeric τ in the CLC (Pritchard et al., 2000; Park et al., 2010) and DnaB (Kim et al., 1996; Gao and McHenry, 2001a).
Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
Bacterial replisomes
2018, Current Opinion in Structural Biology
Citation Excerpt :
A more recent in vitro single-molecule study showed that leading-strand and lagging-strand DNA synthesis by the E. coli replisome can indeed be carried out in a decoupled and stochastic way, in which both polymerases and helicase work independently [36•]. Considering the exchange of active polymerases at replication forks, perhaps new Pol III* can be utilized to synthesize new OFs at, or even behind, the replication fork, as happens with the T7 replisome [27,28•] (Figure 2b). Excess Pol III* can wait or scan for a new primer and start to synthesize an OF once a new primer is available.
Bacterial replisomes are dynamic multiprotein DNA replication machines that are inherently difficult for structural studies. However, breakthroughs continue to come. The structures of Escherichia coli DNA polymerase III (core)–clamp–DNA subcomplexes solved by single-particle cryo-electron microscopy in both polymerization and proofreading modes and the discovery of the stochastic nature of the bacterial replisomes represent notable progress. The structures reveal an intricate interaction network in the polymerase–clamp subassembly, providing insights on how replisomes may work. Meantime, ensemble and single-molecule functional assays and fluorescence microscopy show that the bacterial replisomes can work in a decoupled and uncoordinated way, with polymerases quickly exchanging and both leading-strand and lagging-strand polymerases and the helicase working independently, contradictory to the elegant textbook view of a highly coordinated machine.
Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes
2016, Molecular Cell
Citation Excerpt :
In the absence of competition, polymerases can continually sample all these sites at the replication fork providing multiple points of contact, ensuring a stable attachment. However, under conditions of competition, the relatively low affinity of the individual interactions within the replisome allows polymerases from solution to quickly outcompete those at the fork, driving polymerase exchange and release (Åberg et al., 2016; Geertsema and van Oijen, 2013). Based on our observations, we envision a spectrum of exchange frequencies and coordination mechanisms among replication systems.
The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.
Binding affinities among DNA helicase-primase, DNA polymerase, and replication intermediates in the replisome of bacteriophage T7
2016, Journal of Biological Chemistry
The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase·DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.

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View full text

A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed

Highlights

Section snippets

References and recommended reading

Acknowledgements

Nature

J Biol Chem

Curr Opin Chem Biol

Annu Rev Biophys

Mol Cell

Proc Natl Acad Sci U S A

Proc Natl Acad Sci U S A

Proc Natl Acad Sci U S A

Science

Science

Mol Cell

Nat Struct Mol Biol

Science

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

Nat Rev Microbiol

Biological mechanisms, one molecule at a time

Genes Dev

Single molecule methods with applications in living cells

Curr Opin Biotechnol

Motors, switches, and contacts in the replisome

Annu Rev Biochem

Replisome-mediated DNA replication

Annu Rev Biochem