Creating localized DNA double-strand breaks with microirradiation

Keiji Suzuki, Motohiro Yamauchi, Yasuyoshi Oka, Masatoshi Suzuki, Shunichi Yamashita

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)134-139
Number of pages6
JournalNature Protocols
Volume6
Issue number2
DOIs
Publication statusPublished - Feb 2011
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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