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

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. We present a novel method generating localized DNA double-strand breaks 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 set-up 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 BrdU 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. * *** 0 J 1 0 0 J 2 0 0 J 3 0 0 J 0 1000 2000 3000 Energy applied γH2AX foci (mean intensity)

A synergistic interaction between HDAC‐ and PARP inhibitors in childhood tumors with chromothripsis

“…BJ [Human fibroblast] (RRID:CVCL_3653) (referred from here as BJ WT), UWB1.289 (RRID:CVCL_B079) and UWB1.289 + BRCA1 (RRID:CVCL_B078) cells were purchased from ATCC. The RPE1 TP53 KO cell line was generated by Thorsten Kolb by CRISPR knock out of the TP53 gene in the hTERT‐RPE1 cell line (RRID:CVCL_4388) (referred from here as RPE1 WT) 20 . All these human cell lines have been authenticated using STR or SNP profiling within the last 3 years.…”