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Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells
13:10

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Published on: September 8, 2010

Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG

David M Weinstock1, Koji Nakanishi, Hildur R Helgadottir

  • 1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Methods in Enzymology
|June 24, 2006
PubMed

Insights

This study details reporter assays to measure DNA double-strand break (DSB) repair pathways like nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA) in mammalian cells.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • DNA damage repair is crucial for maintaining genetic integrity.
  • Errors in DNA repair can lead to mutations, cell death, or cancer.
  • Double-strand breaks (DSBs) are a significant form of DNA damage with various causes.

Purpose of the Study:

  • To describe reporter substrates for assaying DSB repair pathways in mammalian cells.
  • To quantify the contributions of nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA) to DSB repair.
  • To enable assessment of factors influencing DSB repair.

Main Methods:

  • Utilizing reporter substrates in mammalian cells, specifically murine embryonic stem cells.
  • Precisely inducing DSBs using I-SceI endonuclease or RAG recombinase.
  • Quantifying repair pathway contributions via fluorescence and PCR-based assays.

Main Results:

  • Demonstrated the utility of reporter substrates for dissecting DSB repair pathways.
  • Enabled quantification of individual pathway contributions (NHEJ, HR, SSA).
  • Showcased the application of these reporters in various mammalian cell types.

Conclusions:

  • Reporter substrates provide a powerful tool for studying DSB repair mechanisms.
  • These assays facilitate the investigation of genetic and physiological influences on DNA repair.
  • The described methods are broadly applicable for assessing DNA repair in diverse cellular contexts.

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