The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding

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Carol Y Ying1, Jean Gautier

  • 1Integrated Program in Cellular, Molecular, and Biophysical Studies, Hammer Health Sciences Center, Columbia University College of Physicians and Surgeons, New York, NY, 10032, USA.

The EMBO Journal
|December 22, 2005
PubMed

Insights

The minichromosome maintenance (MCM) complex

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Eukaryotic DNA replication relies on six essential minichromosome maintenance (MCM) proteins.
  • The precise role of MCM complex ATPase activity in DNA replication remains unclear.

Purpose of the Study:

  • To investigate the function of MCM complex ATPase activity in DNA replication using a cell-free system.
  • To determine if ATP binding/hydrolysis is essential for MCM complex chromatin loading, pre-replicative complex (pre-RC) assembly, and origin unwinding.

Main Methods:

  • Established a replication-competent cell-free system using purified recombinant Xenopus MCM complexes.
  • Utilized mutational analysis of the Walker A motif in MCM6 and MCM7 within the MCM2-7 complex.
  • Assessed chromatin loading, Cdc45 loading, pre-RC assembly, and DNA replication in MCM-depleted extracts.

Main Results:

  • Recombinant MCM2-7 complex successfully loaded onto chromatin and restored DNA replication in depleted extracts.
  • ATP binding and/or hydrolysis by MCM proteins were found to be dispensable for chromatin loading and pre-RC assembly.
  • ATPase activity was essential for origin unwinding during DNA replication, as an ATPase-deficient mutant failed to support replication.

Conclusions:

  • Recombinant MCM proteins can fulfill all replication roles of native MCM complexes, supporting MCM2-7 as the replicative helicase.
  • MCM ATPase activity is crucial for DNA replication initiation (origin unwinding) but not for initial pre-RC assembly.
  • Mutations in MCM ATPase activity uncouple pre-RC assembly from the DNA replication process.

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