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Related Concept Videos

The Replisome03:01

The Replisome

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 the...
The Replisome03:01

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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.
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Replication in Eukaryotes01:29

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Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase
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Published on: April 29, 2010

Stepwise assembly of the human replicative polymerase holoenzyme.

Mark Hedglin1, Senthil K Perumal, Zhenxin Hu

  • 1Department of Chemistry , The Pennsylvania State University , University Park , United States.

Elife
|April 12, 2013
PubMed
Summary

The clamp loader (RFC) unloads sliding clamps (PCNA) from DNA, preventing buildup and recycling PCNA for efficient DNA replication. This ensures limited PCNA is available for polymerase (polδ) during holoenzyme assembly.

Keywords:
DNA polymerase holoenzyme assemblyHumanclamp loaderclamp loadingclamp unloadingsliding clamp

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Area of Science:

  • Molecular Biology
  • DNA Replication
  • Protein-DNA Interactions

Background:

  • Clamp loaders regulate DNA polymerase holoenzyme assembly.
  • The opposing activities of clamp loaders (loading/unloading) and their regulation are not fully understood.

Purpose of the Study:

  • To investigate the stepwise assembly of the human DNA polymerase holoenzyme.
  • To elucidate the regulation of clamp loader (RFC) and sliding clamp (PCNA) interactions during holoenzyme formation.

Main Methods:

  • Förster Resonance Energy Transfer (FRET) was used to monitor protein-DNA interactions.
  • Studied the assembly process of the human DNA polymerase holoenzyme in vitro.

Main Results:

  • RFC loads PCNA onto primer/template DNA but remains bound.
  • PCNA is unloaded by RFC if not immediately captured by polymerase delta (polδ).
  • Polδ captures PCNA from RFC, allowing RFC dissociation and holoenzyme completion.

Conclusions:

  • RFC's unloading activity prevents free PCNA accumulation on DNA.
  • This recycling mechanism maximizes PCNA utilization and supports continuous DNA replication.
  • Regulated unloading is crucial for efficient holoenzyme assembly and function.