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Creating localized DNA double-strand breaks with microirradiation

Abstract

We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2′-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 μM BrdU for 48–72 h, washed with Ca2+- and Mg2+-free PBS(–), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.

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Figure 1: Use of micropore membrane.
Figure 2: Cells covered by a micropore membrane.
Figure 3: Microirradiation creates localized DSBs and recruits DNA damage checkpoint factors.

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Acknowledgements

We thank W.F. Morgan for his critical reading of the manuscript and for helpful comments. This study was supported in part by the Global Center of Excellence (GCOE) Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

AUTHOR CONTRIBUTIONS

K.S. conceived, designed and conducted the study, and drafted the manuscript. M.Y., Y.O. and M.S. provided reagents, carried out the immunofluorescence study and edited the manuscript. S.Y. supervised the project.

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Correspondence to Keiji Suzuki.

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Suzuki, K., Yamauchi, M., Oka, Y. et al. Creating localized DNA double-strand breaks with microirradiation. Nat Protoc 6, 134–139 (2011). https://doi.org/10.1038/nprot.2010.183

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