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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
PubMed

Insights

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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