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Detection of DNA double‐strand breaks by pulsed‐field gel electrophoresis
Author(s) -
Kawashima Yuri,
Yamaguchi Nahomi,
Teshima Rie,
Narahara Hisashi,
Yamaoka Yoshio,
Anai Hirofumi,
Nishida Yoshihiro,
Hanada Katsuhiro
Publication year - 2017
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12457
Subject(s) - biology , dna , gel electrophoresis , dna replication , microbiology and biotechnology , pulsed field gel electrophoresis , dna damage , replication protein a , dna repair , genetics , gene , dna binding protein , genotype , transcription factor
A DNA double‐strand break (DSB) is one of the most cytotoxic DNA lesions because unrepaired DSBs cause chromosomal aberrations and cell death. Although many physiological DSBs occur at DNA replication sites, the molecular mechanisms underlying this remain poorly understood. There was therefore a need to develop a highly specific method to detect DSB fragments containing DNA replication sites. Here we investigated whether pulsed‐field gel electrophoresis (PFGE) combined with visualization of DNA replication sites by immunoblotting using halogenized deoxyuridines, such as BrdU and IdU, was sufficient for this detection. Our methodology enabled us to reproduce previously reported data. In addition, this methodology was also applied to the detection of bacterial infection‐induced DSBs on human chromosomal DNA. Based on our findings, we propose that this strategy combining PFGE with immunoblot analysis will be applicable to studies analyzing the mechanistic details of DNA repair, the DNA damage response and the activity of DNA‐damaging agents.