γH2AX prefers late replicating metaphase chromosome regions

DNA damage response (DDR) constitutes a protein pathway to handle eukaryotic DNA lesions in the context of chromatin. DDR engages the recruitment of signaling, transducer, effector, chromatin modifiers and remodeling proteins, allowing cell cycle delay, DNA repair or induction of senescence or apoptosis. An early DDR-event includes the epigenetic phosphorylation of the histone variant H2AX on serine 139 of the C-termini, so-called gammaH2AX. GammaH2AX foci detected by immunolabeling on interphase nuclei have been largely studied; nonetheless gammaH2AX signals on mitotic chromosomes are less understood. The CHO9 cell line is a subclone of CHO (Chinese hamster ovary) cells with original and rearranged Z chromosomes originated during cell line transformation. As a result, homologous chromosome regions have been relocated in different Z-chromosomes. In a first quantitative analysis of gammaH2AX signals on immunolabeled mitotic chromosomes of cytocentrifuged metaphase spreads, we reported that gammaH2AX¹³⁹ signals of both control and bleomycin-exposed cultures showed statistically equal distribution between CHO9 homologous chromosome regions, suggesting a possible dependence on the structure/function of chromatin. We have also demonstrated that bleomycin-induced gammaH2AX foci map preferentially to DNA replicating domains in CHO9 interphase nuclei. With the aim of understanding the role of gammaH2AX signals on metaphase chromosomes, the relation between 5-ethynyl-2'-deoxyuridine (EdU) labeled replicating chromosome regions and gammaH2AX signals in immunolabeled cytocentrifuged metaphase spreads from control and bleomycin-treated CHO9 cultures was analyzed in the present work. A quantitative analysis of colocalization between EdU and gammaH2AX signals based on the calculation of the Replication Related Damage Distribution Index (RDDI) on confocal metaphase images was performed. RDDI revealed a colocalization between EdU and gammaH2AX signals both in control and bleomycin-treated CHO9 metaphases, suggesting that replication may be involved in H2AX phosphorylation. The possible mechanisms implicated are discussed.