Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks
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Paulina Rybak1, Agnieszka Hoang1, Lukasz Bujnowicz2, Tytus Bernas3, Krzysztof Berniak1, Mirosław Zarębski1, Zbigniew Darzynkiewicz4, Jerzy Dobrucki1
1Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
2Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
3Laboratory for Imaging of Tissue Structure and Function, Nencki Institute of Experimental Biology PAS, Warsaw, Poland
4Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York, USA
Jerzy Dobrucki, email: firstname.lastname@example.org
Keywords: DNA damage, topoisomerase inhibitors, transcription, DNA replication, camptothecin
Received: February 16, 2016 Accepted: June 12, 2016 Published: July 06, 2016
Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.
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