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Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Published on: October 25, 2014
14.9K
Origin recognition complex harbors an intrinsic nucleosome remodeling activity.
Sai Li1, Michael R Wasserman1, Olga Yurieva2
1Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY 10065.
Summary
The origin recognition complex (ORC) remodels chromatin by ejecting specific histone dimers from nucleosomes, ensuring DNA replication origins are accessible. This ATP-dependent process is crucial for efficient origin licensing and activity.
Area of Science:
- Molecular Biology
- Chromatin Biology
- DNA Replication
Background:
- Eukaryotic DNA replication initiates at origins recognized by the origin recognition complex (ORC).
- Nucleosomes can impede ORC binding and MCM helicase loading at replication origins.
- Autonomously replicating sequences (ARSs) are largely nucleosome-depleted but can still be inhibited.
Purpose of the Study:
- To investigate the mechanism by which ORC interacts with and potentially remodels nucleosomes at replication origins.
- To determine if ORC can overcome nucleosome-mediated inhibition of origin licensing.
Main Methods:
- Single-molecule visualization techniques using purified proteins.
- Biochemical assays to study histone eviction and DNA binding.
Main Results:
- ORC actively ejects histone H2A-H2B dimers from nucleosomes in an ATP-dependent manner.
- ORC preferentially evicts canonical H2A over the H2A.Z variant.
- The Orc1 subunit's bromo-adjacent homology (BAH) domain is essential for histone eviction.
Conclusions:
- ORC functions as a nucleosome remodeler, facilitating origin accessibility.
- Histone eviction by ORC is a key step in licensing DNA replication origins.
- The findings reveal a novel role for ORC in chromatin regulation during DNA replication.
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