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Immunofluorescence microscopy of γH2AX and 53BP1 for analyzing the formation and repair of DNA double-strand breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylat...

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Bibliographic Details
Main Authors: Popp, Henning (Author) , Brendel, Susanne (Author) , Hofmann, Wolf-Karsten (Author) , Fabarius, Alice (Author)
Format: Article (Journal) Video
Language:English
Published: 2017
In: JoVE. Video journal
Year: 2017, Issue: 129
ISSN:1940-087X
DOI:10.3791/56617
Subjects:
Film
Online Access:Verlag, Volltext: http://dx.doi.org/10.3791/56617
Verlag, Volltext: https://www.jove.com/video/56617/immunofluorescence-microscopy-h2ax-53bp1-for-analyzing-formation
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Author Notes:Henning D. Popp, Susanne Brendel, Wolf-Karsten Hofmann, Alice Fabarius
Description
Summary:DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of γH2AX and 53BP1, the combined analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on γH2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the γH2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of γH2AX and 53BP1.
Item Description:Enthält auch Textversion
Gesehen am 15.11.2018
Wissenschaftlicher Film. Deutschland. 2017
Physical Description:Online Resource
ISSN:1940-087X
DOI:10.3791/56617

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