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Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Published on: October 25, 2014
Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response
Manolis Papamichos-Chronakis1, Jocelyn E Krebs, Craig L Peterson
1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Abstract:
Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. Here we show that Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA double-strand break (DSB). The failure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability to maintain high levels of histone H2AX phosphorylation and an increased incorporation of the Htz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation and checkpoint adaptation. We propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation.
Insights
Ino80 and Swr1 chromatin remodelers regulate cell cycle adaptation after DNA double-strand breaks (DSBs). Ino80 promotes arrest, while Swr1 facilitates adaptation by controlling histone variant incorporation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes.
- These enzymes play roles in DNA repair processes.
- Cell cycle checkpoint adaptation is crucial for maintaining genomic stability after DNA damage.
Purpose of the Study:
- To investigate the role of Ino80 and Swr1 in cell cycle checkpoint adaptation following DNA double-strand breaks (DSBs).
- To elucidate the mechanism by which these remodelers influence checkpoint adaptation.
- To understand the interplay between Ino80, Swr1, and histone variants in DNA repair.
Main Methods:
- Utilized yeast models to study DNA double-strand break repair.
- Assessed cell cycle checkpoint adaptation in cells lacking Ino80 or Swr1.
- Monitored histone H2AX phosphorylation levels.
- Quantified Htz1p histone variant incorporation into chromatin near DSBs.
Main Results:
- Ino80 is essential for cell cycle checkpoint adaptation after DSBs.
- Loss of Ino80 leads to sustained checkpoint arrest, reduced H2AX phosphorylation, and increased Htz1p incorporation.
- Inactivation of Swr1 reverses these effects, restoring H2AX phosphorylation and checkpoint adaptation.
- Swr1 antagonizes Ino80's role in regulating Htz1p incorporation.
Conclusions:
- Ino80 and Swr1 act antagonistically at DSB sites.
- They regulate the incorporation of distinct histone H2A variants.
- This regulation determines whether cells adapt to or remain arrested by the checkpoint.
- The balance of Ino80 and Swr1 activity is critical for proper cell cycle response to DNA damage.
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