Functional relevance of the histone γH2Ax in the response to DNA damaging agents

Abstract

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays, etoposide, temozolamide, and endogenously generated reactive oxygen species, each of which can include double-strand breaks among their spectra of DNA lesions. The absence or mutation of H2Ax had no influence on sensitivity to cisplatin or mitomycin C. Although UV light induced the highest levels of γH2Ax, mutation of S139 had no influence on UV sensitivity or the UV DNA damage response. Complete loss of H2Ax reduced the survival of cells exposed to UV light and reduced pChk1 induction, suggesting that sites other than S139 may impact the ATR-pChk1 pathway. The relative intensity of γH2Ax measured in Western blots in wild-type cells did not correlate with the functional importance of γH2Ax. The use of γH2Ax as a general biomarker of DNA damage is therefore potentially misleading because it is not an unambiguous indicator of double-strand breaks, and a significant fraction of DNA repair, especially involving nucleotide excision or crosslink repair, can occur without functional involvement of γH2Ax.alkylation | rotenone | caffeine | poly(ADP-ribose) polymerase | veliparib

Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies

“…This event could be a primary stress response, which might attract other proteins involved in DDR-related pathways. Radiation can induce multiple DNA-damage-response pathways, 30 which could explain the fact that we observed cell cycle-independent coilin recruitment at DNA lesions.…”

ATR suppresses apoptosis after UVB light by controlling both translesion synthesis and alternative tolerance pathways

“…However, following UV irradiation, a pan-nuclear cH2AX staining was observed, rather than the discrete foci observed after ionizing radiation. In fact, this early marker does not correspond to DSBs after UV irradiation (de Feraudy et al, 2010), and its role is still not clear given that knocking down H2AX does not affect UV sensitivity (Revet et al, 2011). However, this chromatin remodeling histone can be used as a marker of DNA damage responses.…”

Phosphorylated H2Ax is not an unambiguous marker for DNA double-strand breaks

“…Not all γH2Ax represents DSBs. 5,6 This has become increasingly clear through recent work using UV light that questions several assumptions concerning DNA damage responses, 5,6 First is the realization that the term "DNA Damage Response (DDR)" has been dominated by the DSB scenario; in contrast, a DDR from UV and other agents involves different combinations of players and pathways. Consequently the term DDR should be modified by reference to the agent involved.…”

“…5 In our recent work we further addressed the question of the functional significance of S139 phosphorylation by making an S139A site-directed mutation and inserting it into an H2Ax knockout cell line. 6 The S139A mutant cells were sensitive to killing by γ-rays and reactive oxygen, but only γ-rays induced significant levels of γH2Ax. Strikingly, although in normal cells the S139 site is more highly phosphorylated after UV irradiation than after γ-rays, its absence had no impact on the sensitivity of cells to killing by UV light, cisplatin or DNA-DNA crosslinks!…”