Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA–PK

Abstract

DNA double-strand breaks (DSB) are considered as the most deleterious DNA lesions, and their repair is further complicated by increasing damage complexity. However, the molecular effects of clustered lesions are yet not fully understood. As the locally restricted phosphorylation of H2AX to form γH2AX is a key step in facilitating efficient DSB repair, we investigated this process after localized induction of clustered damage by ionizing radiation. We show that in addition to foci at damaged sites, H2AX is also phosphorylated in undamaged chromatin over the whole-cell nucleus in human and rodent cells, but this is not related to apoptosis. This pan-nuclear γH2AX is mediated by the kinases ataxia telangiectasia mutated and DNA-dependent protein kinase (DNA–PK) that also phosphorylate H2AX at DSBs. The pan-nuclear response is dependent on the amount of DNA damage and is transient even under conditions of impaired DSB repair. Using fluorescence recovery after photobleaching (FRAP), we found that MDC1, but not 53BP1, binds to the nuclear-wide γH2AX. Consequently, the accumulation of MDC1 at DSBs is reduced. Altogether, we show that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and H2AX phosphorylation, yet without eliciting a full pan-nuclear DNA damage response.

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

“…S1D), similar to what had been observed following exposure to bleomycin. Unlike cells showing clear repair foci, where γH2AX and 53BP1 mostly colocalize, 53BP1 did not stain in γH2AX pan-stained cells, which is consistent with previous observations (30,32,33). For serotypes 19F and 3, there was a greater frequency of γH2AX-positive cells compared with uninfected cells, although the ability of these serotypes to induce DSB foci was clearly reduced compared with serotype 4 (Fig.…”

“…Furthermore, in addition to γH2AX appearing as punctate foci in bleomycin-exposed cells, we also observed nuclei with nearly uniform staining for γH2AX (Fig. S1C), which is consistent with studies showing that exposure to high levels of a DNA-damaging agent can result in nuclear-wide staining of γH2AX (pan-γH2AX) (30).…”

“…In contrast to the experiments at MOI of 30-50, most cells that were positive for γH2AX were pan-stained (Fig. 1C), suggesting that, at higher MOI, elevated levels of DNA damage were induced (30). As an alternative approach, we also analyzed the levels of H2AX phosphorylation by Western.…”

“…The pan-γH2AX staining has been reported to occur in human fibroblast cells subjected to Adeno-associated virus (58,60), Chlamydia (33), and UV and ionizing radiation (30,61,62). Recently, such nuclear-wide γH2AX has been shown to occur in highly DNAdamaged cells and is mediated by ATM kinase (30). Here, we found that most of the pan-γH2AX phosphorylation was dependent on ATM kinase and hence constituted part of the DNA damage response cascade induced by S. pneumoniae.…”

223Ra and other bone-targeting radiopharmaceuticals—the translation of radiation biology into clinical practice

“…11 High LET radiation has also been shown to cause not only foci of gH2AX but also a lower intensity, pannuclear background phosphorylation of H2AX in chromatin that has not suffered a DSB after some part of the DNA within the same nucleus has suffered such a break. 12 A similar finding has been observed in human lymphocytes; namely, high LET radiation induces a pan-nuclear signal in addition to discrete foci of gH2AX with only discrete foci being seen following low LET radiation as shown in Figure 1 (S Horn, Queen's University Belfast, 2014, personal communication).…”