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Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click

Hong Zhao1, Jurek Dobrucki, Paulina Rybak

  • 1Department of Pathology, New York Medical College, Valhalla, NY 10595, USA.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|September 10, 2011
PubMed

Insights

Hydrogen peroxide (H2O2) triggers DNA damage response pathways. This study links H2O2-induced DNA damage response events, including ATM activation and γH2AX induction, directly to DNA replication in A549 cells.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Oxidative stress from hydrogen peroxide (H2O2) activates DNA damage response (DDR) pathways.
  • Early DDR events include Ataxia Telangiectasia Mutated (ATM) protein kinase activation and γH2AX induction.
  • In A549 cells, these events predominantly occur during S-phase, coinciding with DNA replication.

Purpose of the Study:

  • To investigate the correlation between H2O2-induced DDR events and DNA replication.
  • To determine if ATM activation and γH2AX induction are linked to active DNA synthesis.
  • To examine the spatial relationship between DNA replication sites and H2AX phosphorylation.

Main Methods:

  • Utilized 5-ethynyl-2'-deoxyuridine (EdU) labeling and click chemistry to identify replicating DNA.
  • Exposed A549 cells to H2O2 after EdU pulse-labeling.
  • Employed multiparameter laser scanning cytometry and confocal microscopy to analyze DDR events and replication.
  • Correlated H2O2-induced ATM activation and γH2AX with DNA replication on a single-cell level.
  • Examined colocalization of replication factories and γH2AX foci.

Main Results:

  • Direct correlation established between H2O2-induced ATM activation, γH2AX induction, and ongoing DNA replication.
  • Confocal microscopy revealed a close association between DNA replication sites and γH2AX foci.
  • Data suggest DDR events are triggered by stalled replication forks or DNA double-strand breaks at H2O2-induced lesions.

Conclusions:

  • DNA replication is closely associated with H2AX phosphorylation in A549 cells following H2O2 exposure.
  • H2O2-induced DDR events may be initiated by replication fork stalling.
  • These findings highlight the interplay between DNA replication stress and the DDR machinery.

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