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Related Experiment Videos

MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg

Melissa C Edwards1, Antonin V Tutter, Christin Cvetic

  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Journal of Biological Chemistry
|June 28, 2002
PubMed
Summary

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The MCM2-7 complex, a key DNA helicase, is recruited to origins of replication. Multiple MCM complexes bind DNA around the origin recognition complex (ORC), with most ready for initiation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The MCM2-7 complex functions as the eukaryotic replicative DNA helicase.
  • Chromatin recruitment involves ORC, Cdc6, and Cdt1; activation requires Cdc45, Cdc7, and Cdk2.
  • A discrepancy exists between the number of MCM and ORC complexes bound to chromatin.

Purpose of the Study:

  • To investigate the mechanism behind the high MCM2-7 to ORC ratio at replication origins.
  • To understand the distribution and function of MCM complexes on DNA.

Main Methods:

  • Analysis of protein binding to immobilized linear DNA fragments using Xenopus egg extracts.
  • Examination of MCM2-7 and ORC complex stoichiometry on DNA fragments of varying lengths.
  • Assessing the recruitment of Cdc45 and phosphorylation status of MCM complexes.

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Main Results:

  • A minimum of 80 bp DNA was required for ORC and MCM2-7 recruitment, initially at a 1:1 ratio.
  • Longer DNA fragments led to a significant increase in the MCM2-7:ORC ratio, indicating MCM distribution.
  • Only a subset of MCM complexes recruited Cdc45, but all showed Cdc7-dependent phosphorylation and potential for Cdk2-dependent Cdc45 loading.

Conclusions:

  • Replication origins in Xenopus egg extracts contain multiple, distributed MCM2-7 complexes.
  • These distributed MCM complexes are initiation-competent, suggesting a broader role than previously thought.
  • The data resolves the paradox of high MCM:ORC ratios by demonstrating MCM complex distribution.