A versatile system to introduce clusters of genomic double-strand breaks in large cell populations

Thorsten Kolb, Umar Khalid, Milena Simović, Manasi Ratnaparkhe, John Wong, Anna Jauch, Peter Schmezer, Agata Rode, Shulamit Sebban, Daniel Haag, Michaela Hergt, Frauke Devens, Yosef Buganim, Marc Zapatka, Peter Lichter, Aurélie Ernst*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Original languageAmerican English
Pages (from-to)303-313
Number of pages11
JournalGenes Chromosomes and Cancer
Volume60
Issue number5
DOIs
StatePublished - May 2021

Bibliographical note

Publisher Copyright:
© 2020 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.

Keywords

  • DNA damage
  • chromothripsis
  • genomic rearrangements

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