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Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells
11:42

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Published on: May 10, 2019

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease

A Richter1, M Baack, H P Holthoff

  • 1Department of Biology, Universität Konstanz, Germany.

Biological Chemistry
|October 29, 1998
PubMed

Insights

Minichromosome maintenance (MCM) proteins bind to open chromatin regions. ATP stabilizes MCM protein interactions, and micrococcal nuclease releases these proteins early during digestion.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Chromatin Biology

Background:

  • Minichromosome maintenance (MCM) proteins are essential for eukaryotic DNA replication initiation.
  • Previous studies required low ionic strength to maintain MCM protein binding to chromatin.
  • ATP is known to stabilize MCM protein-chromatin interactions.

Purpose of the Study:

  • To investigate the binding of human MCM proteins to HeLa cell chromatin under physiological salt conditions.
  • To determine the chromatin accessibility of MCM proteins using micrococcal nuclease digestion.

Main Methods:

  • Chromatin preparation at physiological salt concentrations (100 mM NaCl).
  • Micrococcal nuclease digestion of chromatin.
  • Glycerol gradient sedimentation of released MCM proteins.

Main Results:

  • MCM proteins were released early during micrococcal nuclease digestion.
  • This suggests MCM proteins associate with nuclease-accessible chromatin regions.
  • Released MCM proteins formed a multiprotein complex containing multiple human MCM proteins.

Conclusions:

  • MCM proteins bind to more open chromatin structures.
  • ATP-stabilized MCM-chromatin interactions are accessible to micrococcal nuclease.
  • Human MCM proteins exist as stable multiprotein complexes on chromatin.

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