Replisome mechanics: insights into a twin DNA polymerase machine

doi:10.1016/j.tim.2007.02.007

Trends in Microbiology

Volume 15, Issue 4, April 2007, Pages 156-164

https://doi.org/10.1016/j.tim.2007.02.007 Get rights and content

Chromosomal replicases are multicomponent machines that copy DNA with remarkable speed and processivity. The organization of the replisome reveals a twin DNA polymerase design ideally suited for concurrent synthesis of leading and lagging strands. Recent structural and biochemical studies of Escherichia coli and eukaryotic replication components provide intricate details of the organization and inner workings of cellular replicases. In particular, studies of sliding clamps and clamp-loader subunits elucidate the mechanisms of replisome processivity and lagging strand synthesis. These studies demonstrate close similarities between the bacterial and eukaryotic replication machineries.

Introduction

Replication is carried out in a semiconservative manner as each DNA strand is copied in a 5′ to 3′ direction to create two newly formed daughter strands [1]. The antiparallel conformation of DNA therefore presents major organizational challenges for the replication machinery as the replisome moves in a unidirectional fashion along the separated single strands. These challenges are overcome by the evolution of a dynamic multicomponent holoenzyme complex that employs a dual DNA polymerase design to carry out concurrent continuous and discontinuous synthesis of leading and lagging strands, respectively.

Much insight into how replicases carry out leading and lagging strand synthesis is provided by recent biochemical and structural studies of replication components from bacteria, eukaryotes, archaea and bacteriophage T4 2, 3, 4, 5. These studies include detailed biochemical and structural analyses of sliding clamps, which reveal ring-shaped proteins that confer speed and processivity to the replisome by tethering DNA polymerases to their respective templates. Studies of bacterial and eukaryotic multisubunit clamp loaders elucidate the mechanisms by which these dynamic machines use the energy of ATP to assemble sliding clamps around duplex DNA at primed sites [5]. Biochemical analyses of the Escherichia coli replication fork further characterize the clamp loader as a protein trafficking component that is essential for lagging strand synthesis and the overall organization of the replication machinery 2, 3. Last, biochemical studies characterize the E. coli replicative helicase, which uses the energy of ATP to unwind duplex DNA ahead of the replication fork 6, 7. Owing to space limitations, we focus our attention on the well-defined replication system of E. coli and present certain parallel findings from the eukaryotic system. The brevity of this review does not permit a detailed discussion of replication initiation, which is reviewed elsewhere (see Refs 1, 8, 9, 10).

Section snippets

Mechanics of leading and lagging strand synthesis

Cellular replicases move along DNA in a unidirectional manner while copying both strands of the double helix. The antiparallel conformation of DNA therefore raises questions as to how the replication machinery simultaneously copies both strands of DNA since the direction of nucleic acid synthesis can only occur in a 5′ to 3′ direction. Biochemical studies indicate that cellular replicases overcome this dilemma by synthesizing leading and lagging strands in a continuous and discontinuous manner,

Replisome architecture

Evidence as to how replicases employ clamps and clamp loaders to carry out concurrent leading and lagging strand synthesis efficiently is mainly derived from biochemical and structural studies of E. coli and bacteriophage T4 replication systems (reviewed in Refs 2, 20). Because our current structural understanding of the E. coli replication fork is more advanced, we base our discussion of replisome architecture on the bacterial system, which is presented in Figure 2. The E. coli replicase, DNA

DNA polymerase III core

As illustrated in Figure 2, the E. coli replication machinery employs twin DNA polymerases for concurrent leading and lagging strand synthesis. The two Pol III cores are linked together by the multisubunit ATPase clamp loader (γ complex; light purple). The Pol III core is a heterotrimer composed of α, ɛ and θ subunits [3]. The α subunit is responsible for DNA polymerase activity 1, 2. The ɛ subunit Exhibits 3′ to 5′ exonuclease activity and functions in proofreading the DNA product to ensure

E. coli and eukaryotic sliding clamps

Sliding clamps are ring-shaped proteins that confer processivity onto the replisome by tethering replicative DNA polymerases to their respective template strands. The tether is achieved as the E. coli β-clamp encircles duplex DNA immediately behind Pol III while binding to the α subunit of the polymerase core [24] (Figure 2). As replication ensues, β slides along DNA while remaining bound to the Pol III core, thereby increasing the speed (1 kb s⁻¹) and processivity (>50 kb) of the Pol III core.

The mechanics of clamp loading

Clamp loaders are heteropentameric complexes that use the energy of ATP to assemble sliding clamps around DNA at primed sites 30, 31. A general overview of the clamp loading process is illustrated in Figure 4. In the presence of ATP, the clamp loader binds to and opens the ring-shaped sliding clamp. The ATP-bound clamp-loader complex binds to a primer-template junction, which stimulates the hydrolysis of ATP. ATP hydrolysis results in dissociation of the clamp loader from DNA. Finally, DNA

The E. coli clamp-loader complex

Insight into the organization and mechanism of the clamp loading process has come from crystallographic and biochemical studies of the E. coli γ-complex clamp loader. The ‘minimal’ clamp loader is composed of five subunits (γ₃,δ,δ′) and is sufficient for clamp assembly [31]. The clamp loader also includes additional subunits (χ,Ψ) that are not required for the clamp loading reaction. These small subunits bind to single-stranded DNA binding protein (SSB), which coats single-stranded DNA, and

The eukaryotic clamp-loader complex

The eukaryotic replication factor C (RFC) clamp loader is structurally and functionally similar to the E. coli γ complex (Figure 5b). The five subunits of RFC are encoded by distinct genes but are homologous to one another [52]. The subunits of RFC belong to the AAA+ family of ATPases, and contain the same chain fold as γ, δ and δ′. Furthermore, the ATP sites of RFC are similarly located at subunit interfaces and each uses an arginine finger contained within an SRC motif. RFC and γ complex,

Replicative helicase

The process of DNA replication requires unwinding of the double helix ahead of the replication fork. This function is performed by the E. coli replicative helicase, DnaB, which converts the energy of ATP into the mechanical work of translocation and strand separation. DnaB is a ring-shaped homohexamer that encircles the lagging strand as it unwinds DNA with a 5′ to 3′ polarity [6] (Figure 2). The connection of the Pol III holoenzyme to DnaB, mediated by τ, greatly stimulates DnaB helicase

Replisome dynamics during fork progression

As described earlier, leading strand synthesis proceeds in a continuous and processive manner. The leading strand polymerase therefore uses only one or a few sliding clamps as it copies the entire leading strand. Lagging strand synthesis, however, occurs in a discontinuous fashion – the lagging strand polymerase is recycled among several clamps to synthesize multiple Okazaki fragments. Protein dynamics during lagging strand synthesis are illustrated in Figure 6.

As described earlier, Okazaki

Concluding remarks and future perspectives

Recent structural and biochemical studies of bacterial and eukaryotic replisome components have significantly advanced our understanding of how cellular replicases perform concurrent leading and lagging strand synthesis. The bacterial replisome uses twin DNA polymerases in which each polymerase performs either leading or lagging strand synthesis. Crystallographic and biochemical studies elucidate the organization of bacterial and eukaryotic sliding clamps and clamp loaders and provide insights

Acknowledgements

The authors are grateful to Chiara Indiani and Francine Katz for providing help with artwork. This work was supported by a grant from the National Institutes of Health (GM38839).

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Trends in Microbiology

ReviewMolecular Machines of the CellReplisome mechanics: insights into a twin DNA polymerase machine

Introduction

Section snippets

Mechanics of leading and lagging strand synthesis

Replisome architecture

DNA polymerase III core

E. coli and eukaryotic sliding clamps

The mechanics of clamp loading

The E. coli clamp-loader complex

The eukaryotic clamp-loader complex

Replicative helicase

Replisome dynamics during fork progression

Concluding remarks and future perspectives

Acknowledgements

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Review
Molecular Machines of the Cell
Replisome mechanics: insights into a twin DNA polymerase machine