Low level phosphorylation of histone H2AX on serine 139 (?H2AX) is not associated with DNA double-strand breaks.

ABSTRACT: Phosphorylation of histone H2AX on serine 139 (?H2AX) is an early step in cellular response to a DNA double-strand break (DSB). ?H2AX 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 ?H2AX 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 ?M camptothecin, 10 ?M etoposide or 0.2 ?M 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 ?H2AX foci induced by these drugs. Also, investigated was spatial association between ?H2AX 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 ?H2AX 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 ?H2AX 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 ?H2AX sites of different sizes and intensities.

SUBMITTER: Rybak P 

PROVIDER: S-EPMC5226530 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature