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Generation and Analysis of dsDNA Breaks for Checkpoint and Repair Studies in Fission Yeast

Rohana Ramalingam1, Matthew J O'Connell2

  • 1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 31, 2021
PubMed

Insights

DNA damage triggers complex checkpoint and repair responses. This study details methods for analyzing early events at double-stranded DNA breaks (DSBs) in fission yeast, applicable to other systems.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • DNA damage activates intricate checkpoint and repair mechanisms involving numerous genes.
  • The fission yeast Schizosaccharomyces pombe is a valuable model organism for studying DNA damage responses.
  • Understanding early events at DNA lesions, especially double-stranded DNA breaks (DSBs), is crucial.

Purpose of the Study:

  • To present methodologies for analyzing early events at DSBs.
  • To investigate protein recruitment to DNA lesions.
  • To provide adaptable protocols for various experimental systems.

Main Methods:

  • Detailed analysis of events occurring directly on DNA at DSBs.
  • Methods for tracking protein assembly and signaling cascades at DNA lesions.
  • Utilizing Schizosaccharomyces pombe as a model system for protocol development.

Main Results:

  • Established robust methods for examining DNA repair and checkpoint activation at DSBs.
  • Demonstrated the utility of S. pombe in dissecting early molecular events.
  • Validated protocols for analyzing protein dynamics at DNA damage sites.

Conclusions:

  • The described methods offer precise tools for studying DNA damage response pathways.
  • These techniques are readily adaptable for research in diverse biological models.
  • Further insights into DNA repair and cell cycle regulation can be gained using these approaches.
Keywords:
Double-stranded DNA break (DSB)End resectionImmunofluorescenceQuantitative PCR (qPCR)Schizosaccharomyces pombe

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