UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS

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2022-07-01 17:54

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Conference: Bucharest University Faculty of Physics 2019 Meeting


Section: Biophysics; Medical Physics


Title:
Monitoring the formation and repair of DNA double-strand breaks after irradiation with 60Co γ-rays


Authors:
Ştefan Adrian MINCIU (1), Tatyana BULANOVA (2), Elena KULIKOVA (2), Marcela Elisabeta BARBINTA-PATRASCU (1)


Affiliation:
(1) University of Bucharest, Faculty of Physics, 405 Atomistilor Str., PO Box MG-11, 077125 Bucharest-Magurele, Romania

(2) Joint Institute for Nuclear Research, Radiation Biology Laboratory, Joliot-Curie, 6, Dubna, 141980 Moscow region, Russia


E-mail
elipatras@gmail.com


Keywords:
DNA, ionizing radiation, double strand-breaks, confocal immunofluorescence, fluorescence microscopy


Abstract:
DNA is the informational biomolecule playing the central role in heredity, and its integrity and maintenance represent key factors for cell health. Among all of the lesions induced by chemical, physical or biological agents in the DNA molecule, the double-strand breaks (DSBs) are the most serious DNA lesions, being lethal to cells. In response to DSBs, the protein histone H2AX is phosphorylated at Serine 139 resulting in discrete nuclear foci (γH2AX foci) detectable by immunofluorescence microscopy. Another important DSB-responsive protein promoting repair of DSB is 53BP1 (p53 binding protein 1). Cells of higher eukaryotes developed different biological DSBs repair mechanisms (homologous recombination repair, classical non-homologous end-joining, alternative end-joining, single-strand annealing). In this work, DSBs were induced by subjecting human fibroblasts to ionizing radiations (60Co γ-rays). There is a close correlation between γH2AX foci and DSB numbers and so between the rate of foci loss and DSB repair. The formation and repair of DSBs was monitored by immunofluorescence microscopy of γH2AX and 53BP1 foci. This study can furthermore offer more information about cell response to induced deleterious lesions and give an overall look upon biological repair mechanisms.


References:

[1] Popp, H. D., Brendel, S., Hofmann, W. K., Fabarius, A. Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks. J. Vis. Exp. (129), e56617, doi:10.3791/56617 (2017).

[2] Emil Mladenov, Simon Magin, Aashish Soni, George Iliakis, Seminars in Cancer Biology 37-38 (2016) 51–64.

[3] Markus Löbrich, Atsushi Shibata, Andrea Beucher, Anna Fisher, Michael Ensminger, Aaron A. Goodarzi, Olivia Barton & Penny A. Jeggo (2010) γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization, Cell Cycle, 9:4, 662-669, DOI: 10.4161/cc.9.4.10764.

[4] Kinner A, Wenqi W, Staudt C, Iliakis G. γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res. Oct 2008; 36(17): 5678–5694.

[5] Lucie Jezkova1, Alla Boreyko, Tatiana Bulanova, Marie Davidkova, Iva Falkova, Stanislav Kozubek, Daniel Depes, Evgeny Krasavin, Elena Kruglyakova, Elena Smirnova, Olga Valentova, Mariia Zadneprianetc, and Martin Falk. Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci 2017. „Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci”. Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, 612 65 Brno, Czech Republic. p. 1-32.



Acknowledgement:
This research was supported by the Summer School 2018 at Joint Institute for Nuclear Research, Radiation Biology Laboratory; supervisors: PhD Tatyana Bulanova and PhD Elena Kulikova.