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H2AFX (H2A histone family, member X) is one of several genes coding for histone H2A. In humans and other eukaryotes, the DNA is wrapped around histone-groups, consisting of core histones H2A, H2B, H3 and H4. Thus, the H2AX contributes to the nucleosome-formation and therefore the structure of DNA.

H2AX becomes phosphorylated on serine 139, then called gamma-H2AX, as a reaction on DNA Double-strand breaks (DSB). The kinases of the PI3-family (Ataxia telangiectasia mutated, ATR and DNA-PKcs) are responsible for this phosphorylation, especially ATM. The modification can happen accidentally during replication fork collapse or in the response to ionizing radiation but also during controlled physiological processes such as V(D)J recombination. Gamma-H2AX is a sensitive target for looking at DSBs in cells. The presence of gamma-H2AX by itself, however, is not the evidence of the DSBs. [1]The role of the phosphorylated form of the histone in DNA repair is under discussion but it is known that because of the modification the DNA becomes less condensed, potentially allowing space for the recruitment of proteins necessary during repair of DSBs. Mutagenesis experiments have shown that the modification is necessary for the proper formation of ionizing radiation induced foci in response to double strand breaks, but is not required for the recruitment of proteins to the site of DSBs.

The role of γH2AX in the DNA Damage Response[edit]

The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin.[2] When a double-strand break occurs in DNA, a sequence of events occurs in which H2AX is altered.

Very early after a double-strand break, a specific protein that interacts with and affects the architecture of chromatin is phosphorylated and then released from the chromatin. This protein, heterochromatin protein 1 (HP1)-beta (CBX1), is bound to histone H3 methylated on lysine 9 (H3K9me). Half-maximum release of HP1-beta from damaged DNA occurs within one second.[3] A dynamic alteration in chromatin structure is triggered by HP1-beta release. This alteration in chromatin structure promotes H2AX phosphorylation by ATM, ATR and DNA-PK,[4] allowing formation of γH2AX (H2AX phosphorylated on serine 139). γH2AX can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute.[2] Chromatin with phosphorylated γH2AX extends to about a million base pairs on each side of a DNA double-strand break.[2]

MDC1 (mediator of DNA damage checkpoint protein 1) then binds to γH2AX and the γH2AX/MDC1 complex then orchestrates further interactions in double-strand break repair.[5] The ubiquitin ligases RNF8 and RNF168 bind to the γH2AX/MDC1 complex, ubiquitylating other chromatin components. This allows the recruitment of BRCA1 and 53BP1 to the long, modified γH2AX/MDC1 chromatin.[5] Other proteins that stably assemble on the extensive γH2AX-modified chromatin are the MRN complex (a protein complex consisting of Mre11, Rad50 and Nbs1), RAD51 and the ATM kinase.[6][7] Further DNA repair components, such as RAD52 and RAD54, rapidly and reversibly interact with the core components stably associated with γH2AX-modified chromatin.[7]

The role of γH2AX in chromatin remodeling[edit]

The packaging of eukaryotic DNA into chromatin presents a barrier to all DNA-based processes that require recruitment of enzymes to their sites of action. To allow DNA repair, the chromatin must be remodeled.

γH2AX, the phosphorylated form of H2AX, is involved in the steps leading to chromatin decondensation after DNA double-strand breaks. γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of ionizing irradiation, RNF8 protein can be detected in association with γH2AX.[8] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4,[9] a component of the nucleosome remodeling and deacetylase complex NuRD.

γH2AX as an assay for double-strand breaks[edit]

An assay for γH2AX generally reflects the presence of double-strand breaks in DNA, though the assay may indicate other minor phenomena as well.[10] On the one hand, overwhelming evidence supports a strong, quantitative correlation between γH2AX foci formation and DNA double-strand break induction following ionizing radiation exposure, based on absolute yields and distributions induced per unit dose.[10] On the other hand, not only the formation of distinct γH2AX foci but also the induction of pan-nuclear γH2AX signals have been reported as a cellular reaction to various stressors other than ionizing radiation.[11] The γH2AX signal is always stronger at DNA double-strand breaks than in undamaged chromatin.[11] γH2AX in undamaged chromatin is thought to possibly be generated via direct phosphorylation of H2AX by activated kinases, most likely diffusing from DNA damage sites.


H2AX has been shown to interact with:


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Further reading[edit]