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Published on: August 2, 2024
Histone H2AX phosphorylation: a marker for DNA damage.
Arishya Sharma1, Kamini Singh, Alexandru Almasan
1Department of Cancer Biology, Lerner Research Institute Cleveland Clinic, Cleveland, OH, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 4, 2012
Summary
The DNA damage response involves detecting DNA breaks using the marker gamma-H2AX (phosphorylated H2AX). This sensitive marker aids in studying DNA repair processes in various cell types.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The DNA damage response (DDR) is crucial for maintaining genomic stability following physiological stress or exposure to genotoxic agents like radiation and chemotherapy.
- Double-stranded breaks (DSBs) are critical DNA lesions that trigger rapid cellular responses.
- Histone H2AX phosphorylation at Serine 139 (γ-H2AX) is a highly sensitive and early marker of DSBs.
Purpose of the Study:
- To describe standard protocols for examining the DNA damage response, focusing on γ-H2AX.
- To detail methods for detecting and characterizing γ-H2AX foci for assessing DNA damage and repair.
- To provide practical laboratory procedures for analyzing DNA damage in hematologic and epithelial cells.
Main Methods:
- Detection of γ-H2AX by immunoblotting and immunostaining.
- Microscopic and flow cytometric analysis of γ-H2AX foci.
- Characterization of foci including kinetics, number, size, and morphology.
Main Results:
- Phosphorylation of H2AX (γ-H2AX) is an abundant and rapid response to DSBs, serving as a sensitive marker for DNA damage.
- γ-H2AX detection via immunoblotting and immunostaining allows for quantitative and qualitative assessment of DNA lesions.
- Analysis of γ-H2AX foci provides insights into the dynamics and extent of DNA repair processes.
Conclusions:
- γ-H2AX is a reliable biomarker for studying DNA damage and repair mechanisms.
- Standardized protocols for γ-H2AX detection are essential for accurate assessment of genotoxic stress responses.
- The described methods are applicable to various experimental systems, including cultured hematologic and epithelial cells.
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