Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle. I. ORGANIZATION OF THE CLAMP LOADER

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The ␤ and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli ␤ clamp is known to function with DNA polymerases II and V, indicating that ␤ also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the ␤ clamp. Thus, ␤ interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of ␤ interactive proteins suggests that the prokaryotic ␤ ring participates in a wide variety of reactions beyond its role in chromosomal replication. PCNA ͉ mismatch repairThe Sliding Clamp in Replication

Section: Pcna and Gp45 Participate In Many Dna Metabolic Processesmentioning

confidence: 99%

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

Saro

O'Donnell

2001

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“…Hence, contains an additional 24-kDa C-terminal domain relative to ␥. This 24-kDa domain, referred to here as c , is essential to E. coli and binds both DnaB helicase and Pol III core (7)(8)(9)(10)(11). As illustrated in Fig.…”

mentioning

confidence: 99%

A peptide switch regulates DNA polymerase processivity

Saro

Georgescu

O'Donnell

2003

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Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment. The Escherichia coli replicase contains a subunit ( ) that promotes separation of polymerase from its clamp on finishing DNA segments. This report reveals the mechanism of this process. We find that binds the C-terminal residues of the DNA polymerase. Surprisingly, this same C-terminal ''tail'' of the polymerase interacts with the ␤ clamp, and competes with ␤ for this sequence. Moreover, acts as a DNA sensor. On binding primed DNA, releases the polymerase tail, allowing polymerase to bind ␤ for processive synthesis. But on sensing the DNA is complete (duplex), sequesters the polymerase tail from ␤, disengaging polymerase from DNA. Therefore, DNA sensing by switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.

“…The subunit is also known, to facilitate transfer of an RNA primer from primase to the clamp loader (22). Furthermore, the subunit is required to stabilize the oligomeric structure of the clamp loader upon which the architecture of the replisome is built (23)(24)(25).…”

mentioning

confidence: 99%

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

Yao

Georgescu

Finkelstein

et al. 2009

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110

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 ...