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Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing

Dirk Remus1, Fabienne Beuron, Gökhan Tolun

  • 1Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD, UK.

Cell
|November 10, 2009
PubMed

Insights

The licensing of DNA replication origins involves loading minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes. This study reveals Mcm2-7 forms double hexamers that slide along DNA, crucial for replication initiation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Eukaryotic DNA replication origin licensing ensures one replication per cell cycle.
  • This process involves loading minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
  • Mcm2-7 forms the inactive core of the replicative DNA helicase within pre-RCs.

Purpose of the Study:

  • To reconstitute and biochemically analyze the loading of Mcm2-7 onto DNA.
  • To elucidate the structural organization of Mcm2-7 during the loading process.
  • To understand the mechanism of Mcm2-7 loading and its implications for DNA replication initiation.

Main Methods:

  • Purification of budding yeast proteins.
  • Biochemical reconstitution assays for Mcm2-7 loading.
  • Electron microscopy for structural analysis.

Main Results:

  • Successfully reconstituted Mcm2-7 loading using purified proteins.
  • Demonstrated cooperative loading of Cdt1*Mcm2-7 heptamers.
  • Revealed the formation of stable, head-to-head Mcm2-7 double hexamers with DNA passing through a central channel.
  • Showed that loaded Mcm2-7 can slide passively along double-stranded DNA.

Conclusions:

  • The study provides a detailed mechanistic understanding of Mcm2-7 loading.
  • The findings clarify the structural basis for inactive helicase loading at replication origins.
  • This work has significant implications for understanding replication origin selection, replisome assembly, and DNA unwinding.

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