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EcoSal Plus

Domain 4:

Synthesis and Processing of Macromolecules

Replisome Dynamics during Chromosome Duplication

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  • Authors: Isabel Kurth1, and Mike O’Donnell2
  • Editor: Susan T. Lovett3
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10065; 2: Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10065; 3: Brandeis University, Waltham, Massachusetts
  • Received 27 May 2008 Accepted 20 August 2008 Published 14 August 2009
  • Address correspondence to Mike O’Donnell odonnel@mail.rockefeller.edu.
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  • Abstract:

    This review describes the components of the replisome and the dynamic process in which they function and interact under normal conditions. It also briefly describes the behavior of the replisome during situations in which normal replication fork movement is disturbed, such as when the replication fork collides with sites of DNA damage. DNA Pol III was isolated first from a polA mutant strain that lacked the relatively abundant DNA Pol I activity. Further biochemical studies, and the use of double mutant strains, revealed Pol III to be the replicative DNA polymerase essential to cell viability. In a replisome, DnaG primase must interact with DnaB for activity, and this constraint ensures that new RNA primers localize to the replication fork. The leading strand polymerase continually synthesizes DNA in the direction of the replication fork, whereas the lagging-strand polymerase synthesizes short, discontinuous Okazaki fragments in the opposite direction. Discontinuous lagging-strand synthesis requires that the polymerase rapidly dissociate from each new completed Okazaki fragment in order to begin the extension of a new RNA primer. Lesion bypass can be thought of as a two-step reaction that starts with the incorporation of a nucleotide opposite the lesion, followed by the extension of the resulting distorted primer terminus. A remarkable property of , and many other eubacterial organisms, is the speed at which it propagates. Rapid cell division requires the presence of an extremely efficient replication machinery for the rapid and faithful duplication of the genome.

  • Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2

Key Concept Ranking

DNA Synthesis
0.567365
Replicative DNA Polymerase
0.44277483
Replication Factor C
0.44121575
DNA Polymerase III
0.43199807
0.567365

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/content/journal/ecosalplus/10.1128/ecosalplus.4.4.2
2009-08-14
2017-06-24

Abstract:

This review describes the components of the replisome and the dynamic process in which they function and interact under normal conditions. It also briefly describes the behavior of the replisome during situations in which normal replication fork movement is disturbed, such as when the replication fork collides with sites of DNA damage. DNA Pol III was isolated first from a polA mutant strain that lacked the relatively abundant DNA Pol I activity. Further biochemical studies, and the use of double mutant strains, revealed Pol III to be the replicative DNA polymerase essential to cell viability. In a replisome, DnaG primase must interact with DnaB for activity, and this constraint ensures that new RNA primers localize to the replication fork. The leading strand polymerase continually synthesizes DNA in the direction of the replication fork, whereas the lagging-strand polymerase synthesizes short, discontinuous Okazaki fragments in the opposite direction. Discontinuous lagging-strand synthesis requires that the polymerase rapidly dissociate from each new completed Okazaki fragment in order to begin the extension of a new RNA primer. Lesion bypass can be thought of as a two-step reaction that starts with the incorporation of a nucleotide opposite the lesion, followed by the extension of the resulting distorted primer terminus. A remarkable property of , and many other eubacterial organisms, is the speed at which it propagates. Rapid cell division requires the presence of an extremely efficient replication machinery for the rapid and faithful duplication of the genome.

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Figures

Image of Figure 1
Figure 1

The parental duplex is unwound by the DnaB helicase (yellow) that encircles the lagging strand and travels ahead of the polymerase (blue) in the direction of the moving replication fork. Primase (purple) synthesizes short RNA primers to initiate Okazaki fragment synthesis on the lagging strand. The exposed single lagging-strand template DNA is covered by SSB (pink). The two DNA polymerases are coupled through the clamp loader (green), which uses the energy of ATP hydrolysis to assemble the β processivity clamp (red) around primed sites on the DNA. For simplicity, the χ and ψ subunits of the clamp loader are omitted from the drawing.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 2
Figure 2

Linear N- to C-terminal drawings of the domain architecture of subunits of the replisome are shown to scale, relative to their lengths. Distinct domains are numbered with roman letters, and the amino acid positions listed above the drawings indicate the locations of the first residue and, if the domains are separated by a linker, the last residue of a particular domain. (A) Subunits of Pol III core. Asterisks indicate the locations of the active-site residues (Asp401, Asp403, and Asp555) in the α subunit. L and S indicate the large and small portions of the palm domain. Cter, C-terminal domain; exo, exonuclease subunit. (B) Subunits of the γ complex clamp loader. (C and D) The domain architecture of the β clamp monomer (C) and that of the DnaB helicase and DnaG primase (D) are shown. ssDNA, single-strand DNA.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 3
Figure 3

Shown is a top view of the crystal structure of α, lacking the C-terminal region (residues 918 to 1159) (Protein Data Bank code 2hqa). The active-site residues in the palm domain are indicated by grey spheres.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 4
Figure 4

(A) Ribbon representation of the β homodimer (Protein Data Bank code 2pol). The two monomers (pink and blue) interact head to tail and form a highly symmetrical ring-shaped structure that encircles DNA. The three domains (I, II, and III) of each subunit have identical chain-folding topologies and form the outside perimeter of a continuous antiparallel β sheet. The inside cavity is lined with 12 α helices. (B) Structure of a cocrystal of the β homodimer with a primed DNA template (green). The side view reveals a tilted conformation of the β clamp on DNA, with an angle of approximately 22°. (C) Model of the α subunit of . Pol III bound to the β clamp and DNA.

Adapted from Fig. 7 in ( 26 ) with permission of the publisher.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Figure 5

(A) Schematic representation of the arrangement of the clamp loader subunits demonstrating the circular orientation of the five subunits. The pentameric circular assembly is interrupted by a gap between the δ and δ′ subunits, leaving space for the passage of DNA. The χ and ψ subunits are thought to attach to the γ subunit via ψ. (B) Ribbon representation of the crystal structure of the minimal γ complex clamp loader γδδ′). The C-terminal domains create a tight circular collar. The N termini containing the two AAA+ domains are suspended downward and adapt a conformation in which the δ and δ′ subunits create a gap large enough for the DNA to enter. The β clamp interacts with the N-terminal domains.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 6
Figure 6

(A) ATP binding induces a conformational change in the clamp loader that allows β interaction (step 1). The binding of the β clamp cracks one β dimer interface open, and the β clamp-clamp loader complex gains high affinity for a primer/template junction, allowing the clamp to be placed around primed DNA (step 2). ATP hydrolysis allows the β dimer to close around primed DNA and ejects the clamp loader (step 3). For simplicity, the C-terminal extensions of the τ subunits are not shown.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 7
Figure 7

(A) As the replication fork moves, the DnaB helicase recruits DnaG primase, which synthesizes short RNA primers on the unwound lagging strand. (B) While the lagging-strand polymerase finishes the synthesis of the current Okazaki fragment, the clamp loader displaces primase from the newly synthesized primer and places a β clamp around the primer/template junction. (C) The completion of the Okazaki fragment induces polymerase to dissociate from the β clamp and DNA and allows recruitment to the newly synthesized upstream primer through interaction with the τ subunit of the clamp loader, leaving the β clamp behind. (D) The cycle is complete upon the association of the lagging-strand polymerase with a new β clamp on an upstream RNA primer to begin the synthesis of a new Okazaki fragment.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 8
Figure 8

Lagging-strand polymerase must be able to dissociate from an Okazaki fragment in order to be recycled to new RNA primers during the synthesis of numerous Okazaki fragments. In premature release (left), the polymerase dissociates before finishing the Okazaki fragment, leaving behind a single-strand DNA gap. In collision release (right), the lagging-strand polymerase completes the Okazaki fragment to a nick, and Pol III then disengages from the β clamp. See the text for details.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Image of Figure 9
Figure 9

(A) The two protein binding sites on the two protomers of the β clamp homodimer allow interaction with two DNA polymerases simultaneously. Pol III (blue) retains control of the primer/template during replication under undisturbed conditions. Template lesions (cross) ahead of the polymerase induce Pol III to stall. (B) A TLS polymerase (green) switches places with the stalled Pol III and takes over the primer/template. (C) The TLS polymerase extends the primed site across the lesion. (D) Once the lesion is bypassed, Pol III regains control of the primer/template and continues high-fidelity DNA synthesis.

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Tables

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Table 1

replisome components and associated functions

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2
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Table 2

TLS polymerases

Citation: Kurth I, O’Donnell M. 2009. Replisome Dynamics during Chromosome Duplication, EcoSal Plus 2009; doi:10.1128/ecosalplus.4.4.2

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