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Related Concept Videos

The DNA Replication Fork01:02

The DNA Replication Fork

An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...
The DNA Replication Fork01:02

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An organism’s genome needs to be duplicated in an efficient and error-free manner for its growth and survival. The replication fork is a Y-shaped active region where two strands of DNA are separated and replicated continuously. The coupling of DNA unzipping and complementary strand synthesis is a characteristic feature of a replication fork.   Organisms with small circular DNA, such as E. coli, often have a single origin of replication; therefore, they have only two replication forks, one in...
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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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The Replisome03:01

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Many Proteins Orchestrate Replication at the Origin
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Replication in Eukaryotes02:31

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How a holoenzyme for DNA replication is formed.

Senthil K Perumal1, Wenhui Ren, Tae-Hee Lee

  • 1Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.

Proceedings of the National Academy of Sciences of the United States of America
|December 19, 2012
PubMed
Summary
This summary is machine-generated.

Phage T4 sliding clamp (gp45) loading onto DNA by the gp44/62 clamp loader involves multiple kinetic steps and intermediates. The clamp loader (gp44/62) facilitates holoenzyme formation with polymerase, then dissociates.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The phage T4 DNA replication machinery involves a sliding clamp (gp45) and a clamp loader (gp44/62).
  • Understanding the assembly and disassembly of these protein complexes is crucial for comprehending DNA replication fidelity and dynamics.

Purpose of the Study:

  • To investigate the pathways of phage T4 sliding clamp (gp45) loading and unloading by the gp44/62 clamp loader.
  • To elucidate the mechanism of holoenzyme formation involving gp45, gp44/62, and DNA polymerase.

Main Methods:

  • Utilized Förster resonance energy transfer (FRET)-based single-molecule and ensemble kinetic studies.
  • Analyzed the assembly and disassembly kinetics of protein-DNA complexes.

Main Results:

  • Identified rate-limiting conformational rearrangements during gp45 loading.
  • Revealed that gp45 assembly is a multi-step process, not concerted.
  • Observed two distinct populations of the gp45-gp44/62-DNA complex.
  • Demonstrated ATP hydrolysis-dependent dissociation of gp44/62 during holoenzyme formation.
  • Showed that holoenzyme dissociation from DNA is sensitive to DNA end-blocking, indicating mobility.

Conclusions:

  • The loading of phage T4 sliding clamp (gp45) by the clamp loader (gp44/62) is a complex, multi-step process.
  • The clamp loader plays a critical chaperoning role in recruiting the DNA polymerase for holoenzyme assembly.
  • The dissociation dynamics of the holoenzyme highlight its mobility on DNA.