Role of Histones in DNA Damage
DNA can be damaged by UV-irradiation and drug treatments such as, camptothecin. UV-irradiation is absorbed by pyrimidine bases, thymine and cytosine ultimately resulting in the formation of 6-4 (T-C) photoproduct, this results in a DNA damage response (DDR) causing chromatin decondensation. This results in activation of protein kinases, ATM, ATR and DNA-PKcs at the DNA damage site causing phosphorylation of p53 and histone H2AX. Hundreds to thousands of H2AX molecules are phosphorylated at Ser139 per double-stranded DNA breakage site. This phosphorylation results in the recruitment of DNA damage repair proteins to the site of the DNA damage.
Cell Cycle and DNA Damage Detection via Histone H2AX .
Flow cytometric detection of H2AX can be achieved via tagging the protein with a fluorescently labelled antibody after treatment with BD CytoFix/Perm reagent. Cells can then be labelled with DNA binding dyes such as DAPI (1ug/ml) to show the stage of cell cycle the DNA breakages occur as shown by the presence of H2AX signal. Colorectal carcinoma cell line C80 were treated under normoxic and hypoxic conditions for 3 days with and without drug treatment, see figure.
- Apoptosis, DNA Damage and Cell Proliferation by Becton Dickinson Kit#562253
TS MacFie, R Poulsom, A Parker, G Warnes, TBoitsova, A Nijhuis, N Suraweera, APoehlmann, JSzary, R Feakins, R Jeffery, RW Harper, AM Jubb, JO Lindsay, A Silver. DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the reactive oxygen species H2O2 in active ulcerative colitis and are modulated by 5-aminosalicylic acid. Inflammatory Bowel Disease, Jan 2014.