What does Gamma H2AX do?
This newly phosphorylated protein, gamma-H2AX, is the first step in recruiting and localizing DNA repair proteins. DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents and subsequently, gamma-H2AX foci quickly form.
What happens in the DNA damage response?
The DNA damage response is a network of cellular pathways that sense, signal and repair DNA lesions. Surveillance proteins that monitor DNA integrity can activate cell cycle checkpoints and DNA repair pathways in response to DNA damage, to prevent the generation of potentially deleterious mutations.
What causes DNA damage response?
DDR is a ubiquitous pathway that can be activated by various sources of DNA damage, including ionizing radiation (IR), ultraviolet light, external chemical hazards, oxidative stress, and errors occurring during DNA replication.
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 …
What does comet assay measure?
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.
What are 3 ways DNA gets damaged?
DNA bases can be damaged by: (1) oxidative processes, (2) alkylation of bases, (3) base loss caused by the hydrolysis of bases, (4) bulky adduct formation, (5) DNA crosslinking, and (6) DNA strand breaks, including single and double stranded breaks. An overview of these types of damage are described below.
What prevents DNA damage?
Apoptosis serves as a safeguard mechanism against tumorigenesis. It prevents the increased mutagenesis that excess DNA damage could cause, upon replication. At least 17 DNA repair proteins, distributed among five DNA repair pathways, have a “dual role” in response to DNA damage.
How to assay gamma H2AX?
The detection of γ-H2AX relies on immunological techniques using specific monoclonal and/or polyclonal antibodies against the H2AX C-terminal phosphorylated peptide CKATQAS(PO4)QEY [26]. Total γ-H2AX levels can be measured either in cell and/or tissue lysates or directly in cells and tissues [4].
What is the role of H2AX in DNA repair?
The critical role of H2AX in chromatin reorganization following DNA damage and preventing the separation of DNA ends, thus facilitating DNA end rejoining, can be important not only in the repair of DSBs but also during V(D)J recombination.
What is the role of H2AX in DDR?
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. In general, analysis of H2AX expression can be used to detect the genotoxic effect of different toxic substances.
How is phosphorylation of H2AX mediated by DNA-PK?
DNA-PK mediates phosphorylation of H2AX in cells under hypertonic conditions and during apoptotic DNA fragmentation [28, 29]. However, DNA damage caused by ionizing radiation leads to phosphorylation of H2AX that is mediated by all PIKK kinases, ATM, ATR, and DNA-PK [30].
How are H2AX levels used to diagnose cancer?
When applied to clinical samples from cancer patients, evaluation of H2AX levels may allow not only to monitor the efficiency of anticancer treatment but also to predict of tumor cell sensitivity to DNA damaging anticancer agents and toxicity of anticancer treatment toward normal cells. 1. DNA Double-Strand Breaks
