Characterization of a Triple DNA Polymerase Replisome

McInerney, Peter; Johnson, Aaron; Katz, Francine S.; O'Donnell, Mike

doi:10.1016/j.molcel.2007.06.019

Cited by 171 publications

(186 citation statements)

References 42 publications

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“…The assays of this report are performed at 23°C. E. coli replisomes move Ϸ2-fold faster at 37°C (6,7,9). We would like to note that the level of resolution of the EM-CCD camera is approximately 760 bp/ pixel; therefore, brief pausing events that may occur during replisome translocation would not be detected under our present working conditions.…”

mentioning

confidence: 88%

“…The E. coli replisome consists of the ring shaped hexameric DnaB helicase, DnaG primase, the DNA polymerase III* replicase (Pol III*), and ␤ sliding clamps. Processivity estimates of the E. coli replisome from ensemble studies indicate a lower limit of 50 kb, and an average bulk rate of Ϸ500-700 nt/s at 37°C (6,7). Processivity measurements from other recent single-molecule studies differ over a wide range, from 3 kb to Ͼ80 kb (8,9).…”

mentioning

confidence: 97%

“…The Pol III* assembly contains 2-3 Pol III cores attached to 1 clamp loader apparatus (7). Two Pol III cores within Pol III* are tethered to the leading and lagging strands by ␤ clamps, suggesting that Pol III* tightly adheres to the replication fork for high processivity.…”

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confidence: 99%

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Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

Yao

Georgescu

Finkelstein

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

111

110

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Single-molecule techniques are developed to examine mechanistic features of individual E. coli replisomes during synthesis of long DNA molecules. We find that single replisomes exhibit constant rates of fork movement, but the rates of different replisomes vary over a surprisingly wide range. Interestingly, lagging strand synthesis decreases the rate of the leading strand, suggesting that lagging strand operations exert a drag on replication fork progression. The opposite is true for processivity. The lagging strand significantly increases the processivity of the replisome, possibly reflecting the increased grip to DNA provided by 2 DNA polymerases anchored to sliding clamps on both the leading and lagging strands.polymerase ͉ clamp loader ͉ replicase ͉ sliding clamp ͉ helicase R eplisome machines contain a helicase for DNA unwinding, 2 polymerases for replication of both strands of DNA, and a primase for initiation of lagging strand Okazaki fragments (1-3). Replisomes also contain ring shaped sliding clamp proteins that tether both polymerases to DNA for high processivity [reviewed in ref. 2]. Sliding clamp proteins require a clamp loader machine that couples ATP hydrolysis to open and close clamps onto primed sites. These several components are proposed to function together in a highly coordinated fashion during leading and lagging strand replication (1, 2, 4, 5).The antiparallel structure of DNA requires that 1 strand (the lagging strand) is synthesized as fragments that are extended in the opposite direction of the continuous leading strand. This opposite directionality is resolved by formation of a DNA loop during extension of each Okazaki fragment [i.e., the ''trombone model'' of replication (5)]. Furthermore, each Okazaki fragment requires an RNA primer and assembly of a sliding clamp to initiate chain extension. The repeated initiation events and DNA looping during lagging strand replication may influence the rate and processivity of the replisome.Replisome machines are highly processive entities that synthesize extremely long DNA molecules, making their rate and processivity quite difficult to study by ensemble methods because of the low resolving limit of most gel electrophoretic techniques. Thus, ensemble rate studies can only capture the first 10-20 s of a rapidly moving replisome, and establish a lower limit for processivity. Furthermore, individual replisomes may move forward in an uneven fashion, and this behavior will be masked in an ensemble analysis, which quickly loses synchronicity. Singlemolecule studies circumvent these limitations by real-time observations of individual replication forks over the entire distance of a highly processive binding event. Long DNA products are imaged in the microscope for direct measurements of DNA length.A further advantage to the single-molecule approach is the ability to apply a constant flow of buffer during replication. The use of a flow during replication enables a rigorous test of replisome processivity, as proteins that dissociate from the replisome will ...

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confidence: 88%

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confidence: 97%

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Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

Yao

Georgescu

Finkelstein

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

111

110

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“…1A). Also, studies of the E. coli replisome revealed the presence of a third DNA polymerase, in addition to the canonical leading-and lagging-strand polymerases (22,23).…”

Section: Significancementioning

confidence: 99%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Richardson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

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“…It is an asymmetric dimer or trimer that synthesizes the leading and lagging strands simultaneously at the replication fork 6, 7, 12. A helicase unwinds the double‐stranded DNA (dsDNA) into two anti‐parallel template strands.…”

Section: Introductionmentioning

confidence: 99%

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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Characterization of a Triple DNA Polymerase Replisome

Cited by 171 publications

References 42 publications

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

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