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High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells

Florian J Groelly1, Rebecca A Dagg1, Jonathan Mailler2

  • 1Genome Stability and Tumourigenesis Group, Department of Oncology, Oxford Institute for Radiation Oncology, University of Oxford, Oxford OX3 7DQ, UK.

STAR Protocols
|January 4, 2023
PubMed

Insights

This study introduces a new protocol to map where mitotic DNA synthesis (MiDAS) occurs in cells with DNA replication stress. This method uses EdU labeling and next-generation sequencing for high-resolution genome-wide identification of MiDAS sites.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • DNA replication stress is a critical challenge for cell division.
  • Under-replicated DNA in mitosis triggers specific repair pathways.
  • Mitotic DNA synthesis (MiDAS) is a key mechanism for repairing such DNA damage during mitosis.

Purpose of the Study:

  • To develop and describe a high-resolution, genome-wide protocol for identifying MiDAS events.
  • To pinpoint the specific genomic locations where MiDAS occurs in cells under replication stress.

Main Methods:

  • Utilizing EdU incorporation to label newly synthesized DNA in mitotic cells.
  • Employing next-generation sequencing for high-resolution analysis.
  • Developing a protocol for isolating EdU-labeled DNA from mitotic cells.

Main Results:

  • The protocol enables genome-wide identification of MiDAS sites.
  • High-resolution mapping of where MiDAS occurs is achieved.
  • The method is effective in cells exposed to aphidicolin, a replication inhibitor.

Conclusions:

  • The described protocol provides a powerful tool for studying MiDAS.
  • This method advances our understanding of DNA repair during cell division.
  • It facilitates high-resolution investigation of genome stability maintenance.
Keywords:
Cell BiologyCell cultureChIPseqMolecular BiologySequencing

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