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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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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...
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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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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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Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation.

Daniel Ramírez Montero1, Humberto Sánchez1, Edo van Veen1

  • 1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

Nature Communications
|April 14, 2023
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Summary

The eukaryotic CMG helicase (Cdc45-Mcm2-7-GINS) orchestrates DNA replication. Researchers quantified its single-molecule motion, revealing ATP-dependent unidirectional movement crucial for initiating DNA replication.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • The CMG helicase complex is essential for eukaryotic DNA replication, orchestrating the replisome at replication forks.
  • Previous single-molecule studies of CMG motion relied on pre-formed complexes with unknown assembly mechanisms.

Purpose of the Study:

  • To reconstitute and activate CMG from purified yeast proteins.
  • To quantify the single-molecule motion of reconstituted CMG on DNA.
  • To elucidate the role of nucleotide binding in CMG activation and DNA engagement.

Main Methods:

  • Single-molecule biophysics techniques.
  • Reconstitution of the CMG helicase complex from purified yeast proteins.
  • Biochemical assays to monitor CMG motion and DNA melting.

Main Results:

  • CMG exhibits two modes of motion: unidirectional translocation and diffusion.
  • ATP promotes unidirectional translocation, while its absence favors diffusive motion.
  • Nucleotide binding halts CMG diffusion, independent of DNA melting.

Conclusions:

  • Nucleotide binding is critical for newly assembled CMG to engage DNA, halting diffusion and initiating replication.
  • This study provides a mechanism for CMG activation and engagement at the start of DNA replication.
  • Understanding CMG motion is key to understanding the fundamental process of DNA replication.