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DNA induces conformational changes in a recombinant human minichromosome maintenance complex

Emma L Hesketh1, Richard P Parker-Manuel1, Yuriy Chaban2

  • 1From the Department of Biology, University of York, York YO10 5DD and.

The Journal of Biological Chemistry
|February 5, 2015
PubMed

Insights

Researchers produced functional recombinant human MCM (minichromosome maintenance) complexes, demonstrating their ATP-dependent DNA unwinding and hydrolysis capabilities. This breakthrough enables in vitro studies of the human replicative helicase.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA replication relies on the minichromosome maintenance (MCM) complex, a key component of the replicative helicase.
  • While archaeal and yeast MCM complexes show DNA unwinding activity, higher eukaryotic MCM helicase activity is often studied within larger complexes.
  • The human MCM complex's independent function and structure remained less characterized due to production challenges.

Purpose of the Study:

  • To produce recombinant human MCM (hMCM) complex in Escherichia coli.
  • To characterize the biochemical and structural properties of the recombinant hMCM complex.
  • To assess the functional activity of hMCM in DNA unwinding and ATP hydrolysis.

Main Methods:

  • Recombinant expression of the human MCM complex in E. coli.
  • Biochemical assays to measure ATP hydrolysis and DNA unwinding activity.
  • Single-particle asymmetric electron microscopy (EM) for structural analysis.

Main Results:

  • Successfully produced functional recombinant hMCM complex lacking post-translational modifications.
  • Demonstrated that recombinant hMCM exhibits ATP hydrolysis and duplex DNA unwinding capabilities.
  • Structural analysis revealed that hMCM forms a hexamer and undergoes conformational changes upon DNA binding.

Conclusions:

  • Recombinant hMCM is a functional hexameric DNA helicase.
  • The produced hMCM complex serves as a valuable tool for in vitro biochemical reconstitution of the human replicative helicase.
  • This work facilitates further mechanistic studies of human DNA replication initiation and elongation.
Keywords:
ATPaseDNA HelicaseDNA ReplicationElectron Microscopy (EM)MCMMinichromosome MaintenanceRecombinant Protein Expression

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