H2A histone family member X (usually abbreviated as H2AX) is a type of
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wr ...
protein from the
H2A family encoded by the ''H2AFX'' gene. An important phosphorylated form is γH2AX (S139), which forms when double-strand breaks appear.
In humans and other
eukaryotes
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
, the
DNA is wrapped around
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wr ...
octamers, consisting of
core histones
In biology, histones are highly Base (chemistry), basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosom ...
H2A,
H2B,
H3 and
H4, to form
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
. H2AX contributes to
nucleosome
A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamen ...
-formation, chromatin-remodeling and
DNA repair
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA dam ...
, and is also used ''in vitro'' as an assay for double-strand breaks in
dsDNA.
Formation of γH2AX
H2AX becomes phosphorylated on serine 139, then called γH2AX, as a reaction on
DNA double-strand breaks (DSB). The kinases of the PI3-family (
Ataxia telangiectasia mutated
ATM serine/threonine kinase or Ataxia-telangiectasia mutated, symbol ATM, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the DNA ...
,
ATR ATR may refer to:
Medicine
* Acute transfusion reaction
* Ataxia telangiectasia and Rad3 related, a protein involved in DNA damage repair
Science and mathematics
* Advanced Test Reactor, nuclear research reactor at the Idaho National Laboratory, ...
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. γH2AX is a sensitive target for looking at DSBs in cells. The presence of γH2AX by itself, however, is not the evidence of the DSBs. 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.
Function
DNA damage response
The
histone
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wr ...
variant H2AX constitutes about 2-25% of the H2A histones in mammalian chromatin.
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
The family of heterochromatin protein 1 (HP1) ("Chromobox Homolog", CBX) consists of highly conserved proteins, which have important functions in the cell nucleus. These functions include gene repression by heterochromatin formation, transcrip ...
(HP1)-beta (
CBX1), is bound to
histone H3
Histone H3 is one of the five main histones involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H3 is involved with the structure of the nucleosomes of the 'beads on a stri ...
methylated
In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...
on lysine 9 (H3K9me). Half-maximum release of HP1-beta from damaged DNA occurs within one second.
A dynamic alteration in chromatin structure is triggered by HP1-beta release. This alteration in chromatin structure promotes H2AX phosphorylation by
ATM,
ATR ATR may refer to:
Medicine
* Acute transfusion reaction
* Ataxia telangiectasia and Rad3 related, a protein involved in DNA damage repair
Science and mathematics
* Advanced Test Reactor, nuclear research reactor at the Idaho National Laboratory, ...
and
DNA-PK,
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.
Chromatin with phosphorylated γH2AX extends to about a million base pairs on each side of a DNA double-strand break.
MDC1
Mediator of DNA damage checkpoint protein 1 is a 2080 amino acid long protein that in humans is encoded by the ''MDC1'' gene located on the short arm (p) of chromosome 6. MDC1 protein is a regulator of the Intra-S phase and the G2/M cell cycle chec ...
(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.
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.
Other proteins that stably assemble on the extensive γH2AX-modified chromatin are the
MRN complex
The MRN complex (MRX complex in yeast) is a protein complex consisting of Mre11, Rad50 and Nbs1 (also known as Nibrin in humans and as Xrs2 in yeast). In eukaryotes, the MRN/X complex plays an important role in the initial processing of double-st ...
(a
protein complex
A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multienzyme complexes, in which multiple catalytic domains are found in a single polypeptide chain.
Protein c ...
consisting of
Mre11
Double-strand break repair protein MRE11 is an enzyme that in humans is encoded by the ''MRE11'' gene. The gene has been designated ''MRE11A'' to distinguish it from the pseudogene ''MRE11B'' that is nowadays named ''MRE11P1''.
Function
This ge ...
,
Rad50
DNA repair protein RAD50, also known as RAD50, is a protein that in humans is encoded by the ''RAD50'' gene.
Function
The protein encoded by this gene is highly similar to ''Saccharomyces cerevisiae'' Rad50, a protein involved in DNA double- ...
and
Nbs1
Nibrin, also known as NBN or NBS1, is a protein which in humans is encoded by the ''NBN'' gene.
Function
Nibrin is a protein associated with the repair of double strand breaks (DSBs) which pose serious damage to a genome. It is a 754 amino ac ...
),
RAD51
DNA repair protein RAD51 homolog 1 is a protein encoded by the gene ''RAD51''. The enzyme encoded by this gene is a member of the RAD51 protein family which assists in repair of DNA double strand breaks. RAD51 family members are homologous to th ...
and the
ATM kinase
ATM serine/threonine kinase or Ataxia-telangiectasia mutated, symbol ATM, is a serine/ threonine protein kinase that is recruited and activated by DNA double-strand breaks. It phosphorylates several key proteins that initiate activation of the D ...
.
Further DNA repair components, such as RAD52 and RAD54, rapidly and reversibly interact with the core components stably associated with γH2AX-modified chromatin.
The constitutive level of γH2AX expression in live cells, untreated by exogenous agents, likely represents DNA damage by endogenous oxidants generated during cellular respiration.
In chromatin remodeling
The packaging of eukaryotic DNA into
chromatin
Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...
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 radiation
Ionizing radiation (or ionising radiation), including nuclear radiation, consists of subatomic particles or electromagnetic waves that have sufficient energy to ionize atoms or molecules by detaching electrons from them. Some particles can travel ...
,
RNF8
E3 ubiquitin-protein ligase RNF8 is an enzyme that in humans is encoded by the ''RNF8'' gene. RNF8 has activity both in immune system functions and in DNA repair.
Function
The protein encoded by this gene contains a RING finger motif and an ...
protein can be detected in association with γH2AX.
RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with
CHD4
Chromodomain-helicase-DNA-binding protein 4 is an enzyme that in humans is encoded by the ''CHD4'' gene.
Function
The product of this gene belongs to the SNF2/RAD54 helicase family. It represents the main component of the nucleosome remodeling ...
,
a component of the nucleosome remodeling and deacetylase complex
NuRD
In the field of molecular biology, the Mi-2/NuRD (Nucleosome Remodeling Deacetylase) complex, is a group of associated proteins with both ATP-dependent chromatin remodeling and histone deacetylase activities. , Mi-2/NuRD was the only known prot ...
.
γH2AX as an assay for double-strand breaks
An assay for γH2AX generally reflects the presence of double-strand breaks in DNA, though the assay may indicate other minor phenomena as well.
On the one hand, overwhelming evidence supports a strong, quantitative correlation between γH2AX foci formation and DNA double-strand break induction following
ionizing radiation
Ionizing radiation (or ionising radiation), including nuclear radiation, consists of subatomic particles or electromagnetic waves that have sufficient energy to ionize atoms or molecules by detaching electrons from them. Some particles can travel ...
exposure, based on absolute yields and distributions induced per unit dose.
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.
The γH2AX signal is always stronger at DNA double-strand breaks than in undamaged chromatin.
[ 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. In using γH2AX as a marker for double strand breaks, it is important to recognize that it is a down-stream proxy that can be useful for representing DNA damage repair. It does not represent double strand breaks themselves and this needs careful consideration when interpreting data from such assays.]
Interactions
H2AX has been shown to interact
Advocates for Informed Choice, dba interACT or interACT Advocates for Intersex Youth, is a 501(c)(3) nonprofit organization using innovative strategies to advocate for the legal and human rights of children with intersex traits. The organizati ...
with:
* BARD1
BRCA1-associated RING domain protein 1 is a protein that in humans is encoded by the ''BARD1'' gene. The human BARD1 protein is 777 amino acids long and contains a RING finger domain (residues 46-90), four ankyrin repeats (residues 420-555), and t ...
,[
* ]BRCA1
Breast cancer type 1 susceptibility protein is a protein that in humans is encoded by the ''BRCA1'' () gene. Orthologs are common in other vertebrate species, whereas invertebrate genomes may encode a more distantly related gene. ''BRCA1'' is a h ...
* Bloom syndrome protein
Bloom syndrome protein is a protein that in humans is encoded by the ''BLM'' gene and is not expressed in Bloom syndrome.
The Bloom syndrome gene product is related to the RecQ subset of DExH box-containing DNA helicases and has both DNA-stimula ...
,[
* ]MDC1
Mediator of DNA damage checkpoint protein 1 is a 2080 amino acid long protein that in humans is encoded by the ''MDC1'' gene located on the short arm (p) of chromosome 6. MDC1 protein is a regulator of the Intra-S phase and the G2/M cell cycle chec ...
,
* Nibrin
Nibrin, also known as NBN or NBS1, is a protein which in humans is encoded by the ''NBN'' gene.
Function
Nibrin is a protein associated with the repair of double strand breaks (DSBs) which pose serious damage to a genome. It is a 754 amino ac ...
, and
* TP53BP1
Tumor suppressor p53-binding protein 1 also known as p53-binding protein 1 or 53BP1 is a protein that in humans is encoded by the ''TP53BP1'' gene.
Clinical significance
53BP1 is underexpressed in most cases of triple-negative breast cancer.
D ...
.
References
Further reading
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{{PDB Gallery, geneid=3014
Genes
DNA repair