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Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

Katherine Roper1, Dawn Coverley

  • 1Biology Department, University of York, Heslington, York, YO10 5YW, UK.

Experimental Cell Research
|December 27, 2011
PubMed

Insights

This study introduces a cell-free system to investigate DNA damage response. It shows that damage signals inhibit DNA replication and phosphorylate histone H2AX in intact nuclei, independent of direct DNA lesions.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Mammalian cells possess DNA damage response mechanisms involving cell cycle arrest and repair.
  • DNA damage response (DDR) involves inhibition of DNA replication and phosphorylation of histone H2AX.

Purpose of the Study:

  • To develop and utilize a cell-free system to study the DNA damage response.
  • To investigate the effects of DNA damage signaling on nuclei without direct DNA lesions.
  • To screen for inhibitors of DNA damage response kinases.

Main Methods:

  • A cell-free system using damage-activated cell extracts and naïve nuclei.
  • Treatment of cells or nuclei with etoposide to induce DNA damage.
  • In vitro replication assays and histone H2AX phosphorylation analysis.
  • Screening using wortmannin and LY294002 inhibitors.

Main Results:

  • Cell-free extracts from etoposide-treated cells inhibited DNA replication initiation and elongation in naïve nuclei.
  • Histone H2AX phosphorylation occurred in naïve nuclei, dependent on phosphatidylinositol 3-kinase-like protein kinases.
  • Phosphorylated H2AX was diffuse throughout the nucleus, not focal, in the absence of DNA lesions.
  • The system successfully identified inhibitors of DNA damage response kinases.

Conclusions:

  • A novel cell-free system effectively reconstitutes key aspects of the DNA damage response.
  • DNA damage signaling can inhibit replication and induce histone H2AX phosphorylation in undamaged nuclei.
  • This system provides a valuable platform for screening DNA damage response modulators.

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