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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with...
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Replication in Eukaryotes01:29

Replication in Eukaryotes

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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
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DNA Replication02:40

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DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
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Homologous Recombination02:31

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Related Experiment Video

Updated: Mar 10, 2026

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level
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Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

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How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication.

Joseph T P Yeeles1, Agnieska Janska1, Anne Early1

  • 1The Francis Crick Institute, Clare Hall Laboratory, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.

Molecular Cell
|December 20, 2016
PubMed
Summary

The eukaryotic replisome, a DNA replication machine, was reconstituted with purified proteins. Key factors Mrc1 and Csm3/Tof1 are vital for DNA replication speed, with DNA polymerase ε and PCNA essential for maximal rates.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The eukaryotic replisome coordinates DNA helicase (CMG) with DNA polymerases for DNA replication.
  • Understanding the precise roles of each component is crucial for comprehending DNA synthesis.
  • Replicative stress can impact DNA replication fidelity and speed.

Purpose of the Study:

  • To reconstitute the eukaryotic replisome using purified proteins.
  • To investigate the roles of Mrc1, Csm3/Tof1, and DNA polymerases in replisome progression and DNA synthesis rates.
  • To elucidate the mechanisms underlying leading-strand synthesis and polymerase switching.

Main Methods:

  • Reconstitution of the eukaryotic replisome with purified proteins.
  • In vitro assays to measure CMG assembly and activation.
  • Analysis of replisome progression rates in the presence of various factors and DNA polymerases.

Main Results:

  • Successful reconstitution of the eukaryotic replisome, starting with CMG assembly and activation.
  • Mrc1 and Csm3/Tof1 were identified as crucial for in vivo replisome progression rates.
  • Maximal DNA replication rates were achieved with DNA polymerase ε and PCNA for leading-strand synthesis.
  • DNA polymerase δ plays a role in both lagging-strand synthesis and initiating leading-strand synthesis.
  • Polymerase switching contributes to leading-strand synthesis under replicative stress.

Conclusions:

  • The study successfully reconstituted the eukaryotic replisome, providing insights into its complex coordination.
  • Mrc1 and Csm3/Tof1 are essential regulators of replisome speed.
  • DNA polymerase ε with PCNA drives maximal leading-strand synthesis, while DNA polymerase δ has dual roles.
  • Polymerase switching is a key mechanism for maintaining DNA synthesis fidelity during replicative stress.