What is Gamma-H2AX foci?

When DNA undergoes double-strand break damage, H2AX is phosphorylated by ATM (or ATR). This phosphorylation of H2AX is named gamma-H2AX (y-H2AX). In this regard gamma-H2AX foci should be regarded as a marker for DSBs rather than for repair, although H2AX phosphorylation may promote DSBR.

What is Gamma-H2AX assay?

Since it was first demonstrated by Rogakou, Bonner and colleagues that histone H2AX is rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation [37], the γ-H2AX assay has been widely used as a sensitive molecular marker of DNA damage and DSB repair capacity in a …

How does Gamma-H2AX work?

H2AX phosphorylation creates an epigenetic signal that is recognized by specific domains on downstream DDR proteins. Further, γH2AX facilitates DSB rejoining by anchoring broken ends together through nucleosome repositioning at damaged sites and a reduction in chromatin density.

What is DNA damage foci?

In response to DNA damage, DNA repair foci, which are the subnuclear foci formed by DNA repair factors that accumulate specifically at damaged sites, are detected as distinct spots in nuclei (Rothkamm et al., 2015).

What does Y H2AX do?

Phosphorylation of H2AX plays a key role in DDR and is required for the assembly of DNA repair proteins at the sites containing damaged chromatin as well as for activation of checkpoints proteins which arrest the cell cycle progression.

What are nuclear foci?

The number of γH2AX foci per nucleus is an accepted measure of the number of DNA double-strand breaks in single cells. One of the experimental techniques for γH2AX detection in cultured cells is immunofluorescent labelling of γH2AX and nuclei followed by microscopy imaging and analysis.

When does the number of γH2AX foci peak?

As shown in Fig. 1 (A,C), the number of γH2AX foci reached a peak at 30 min post-IR and then decreased gradually in cells exposed to 1 Gy (low dose). When the cells were irradiated with 4 Gy (high dose), the number of γH2AX foci reached a peak at 1 h and then declined.

How are γH2AX foci formed in HeLa cells?

(A, B) γH2AX foci in HeLa cells after irradiation with 1 Gy (A) or 4 Gy (B) γ-rays. The cells were collected and fixed at the indicated time points after irradiation. (C) The dynamics of γH2AX foci formation after irradiation as shown in (A) & (B).

How are DSBs related to γ-H2AX focus?

Although it is commonly accepted that a γ-H2AX focus indicates the presence of a double strand break (DSB), while foci disappearance is associated with the repair of the DNA damage, the exact relationship between the number of foci and the number of DSBs is still a matter of debate [ 12 – 14 ].

What happens to H2AX foci after ionizing radiation?

To investigate the dynamics of H2AX phosphorylation post-ionizing radiation (IR), HeLa cells were irradiated with 1 Gy (low dose) or 4 Gy (high dose). As shown in Fig. 1 (A,C), the number of γH2AX foci reached a peak at 30 min post-IR and then decreased gradually in cells exposed to 1 Gy (low dose).