Abstract
In eukaryotes, a cascade of events named DNA damage response (DDR) has evolved to handle DNA lesions. DDR engages the recruitment of signaling, checkpoint control, repair and chromatin remodeling protein complexes, allowing cell cycle delay, DNA repair or induction of apoptosis. An early DDR event involves the phosphorylation of the histone variant γH2AX on serine 139 (H2AX139 phosphorylation) originating the so-called γH2AX. DDR-related H2AX139 phosphorylation have been extensively studied in interphase nuclei. More recently, γH2AX signals on mitotic chromosomes of asynchronously growing cell cultures were observed. We performed a quantitative analysis of γH2AX signals on γH2AX immunolabeled cytocentrifuged metaphase spreads, analyzing the γH2AX signal distributions of CHO9 chromosomes harboring homologous regions both in control and bleomycin (BLM)-treated cultures. We detected γH2AX signals in CHO9 chromosomes of controls which significantly increase after BLM-exposure. γH2AX signals were uniformly distributed in chromosomes of controls. However, the γH2AX signal distribution in BLM exposed cells was significantly different between chromosomes and among chromosome regions, with few signals near the centromeres and a tendency to increase towards the telomeres. Interestingly, both basal and BLM-induced γH2AX signal distribution were statistically equal between CHO9 homologous chromosome regions. Our results suggest that BLM exerts an effect on H2AX139 phosphorylation, prevailing towards acetylated and gene-rich distal chromosome segments. The comparable H2AX139 phosphorylation of homologous regions puts forward its dependence on chromatin structure or function and its independence of the position in the karyotype.
Highlights
DNA double-strand breaks (DSB) involve special challenges for the cells
The regions of γH2AX signals observed in metaphase chromosomes were generally large, involving several chromosome bands
ΓH2AX signals were detected in a high percentage of control metaphases, probably involving endogenous DNA damage or tensional stress
Summary
DNA double-strand breaks (DSB) involve special challenges for the cells. Persisting DSB may lead to chromosome fragments loss during anaphase originating micronuclei. Unrepaired or mis-repaired DSB could lead to genomic or chromosomal instability, senescence, malignant transformation or cell death [1] [2]. A hierarchical and timely regulated cascade of events called DNA damage response (DDR) has evolved which includes the recruitment of signaling, checkpoint control, DNA repair and chromatin remodeling protein complexes. DDR allows cell cycle delay, DNA repair or induce cell dead by recruiting proteins of the apoptotic cascade [3]. An early DDR event engages the phosphorylation of the histone variant H2AX involving the oxygen in the γ-position of serine 139 in the C-terminal consensus serine-glutamine (SQ) motif (H2AX139 phosphorylation) giving rise to the named γH2AX [4]
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