H2AFX

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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.

Interactions[edit]

H2AX has been shown to interact with:

References[edit]

  1. ^ Rybak P, Hoang A, Bujnowicz L, Bernas T, Berniak K, Zarębski M, Darzynkiewicz Z, Dobrucki J. (2016) Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks. Oncotarget. 7:49574-49587. PMID 27391338.
  2. ^ a b c Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998). "DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139". J. Biol. Chem. 273 (10): 5858–68. doi:10.1074/jbc.273.10.5858. PMID 9488723. 
  3. ^ Ayoub N, Jeyasekharan AD, Bernal JA, Venkitaraman AR (2008). "HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response". Nature. 453 (7195): 682–6. doi:10.1038/nature06875. PMID 18438399. 
  4. ^ Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y (2003). "Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes". J. Biol. Chem. 278 (22): 20303–12. doi:10.1074/jbc.M300198200. PMID 12660252. 
  5. ^ a b Scully R, Xie A (2013). "Double strand break repair functions of histone H2AX". Mutat. Res. 750 (1-2): 5–14. doi:10.1016/j.mrfmmm.2013.07.007. PMC 3818383Freely accessible. PMID 23916969. 
  6. ^ Bekker-Jensen S, Lukas C, Kitagawa R, Melander F, Kastan MB, Bartek J, Lukas J (2006). "Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks". J. Cell Biol. 173 (2): 195–206. doi:10.1083/jcb.200510130. PMC 2063811Freely accessible. PMID 16618811. 
  7. ^ a b Essers J, Houtsmuller AB, van Veelen L, Paulusma C, Nigg AL, Pastink A, Vermeulen W, Hoeijmakers JH, Kanaar R (2002). "Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage". EMBO J. 21 (8): 2030–7. doi:10.1093/emboj/21.8.2030. PMC 125370Freely accessible. PMID 11953322. 
  8. ^ Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, Lukas J (2007). "RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins". Cell. 131 (5): 887–900. doi:10.1016/j.cell.2007.09.040. PMID 18001824. 
  9. ^ Luijsterburg MS, Acs K, Ackermann L, Wiegant WW, Bekker-Jensen S, Larsen DH, Khanna KK, van Attikum H, Mailand N, Dantuma NP (2012). "A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure". EMBO J. 31 (11): 2511–27. doi:10.1038/emboj.2012.104. PMC 3365417Freely accessible. PMID 22531782. 
  10. ^ a b Rothkamm K, Barnard S, Moquet J, Ellender M, Rana Z, Burdak-Rothkamm S (2015). "DNA damage foci: Meaning and significance". Environ. Mol. Mutagen. 56 (6): 491–504. doi:10.1002/em.21944. PMID 25773265. 
  11. ^ a b Meyer B, Voss KO, Tobias F, Jakob B, Durante M, Taucher-Scholz G (2013). "Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK". Nucleic Acids Res. 41 (12): 6109–18. doi:10.1093/nar/gkt304. PMC 3695524Freely accessible. PMID 23620287. 
  12. ^ a b Mallery DL, Vandenberg CJ, Hiom K (Dec 2002). "Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains". The EMBO Journal. 21 (24): 6755–62. doi:10.1093/emboj/cdf691. PMC 139111Freely accessible. PMID 12485996. 
  13. ^ a b Chen A, Kleiman FE, Manley JL, Ouchi T, Pan ZQ (Jun 2002). "Autoubiquitination of the BRCA1*BARD1 RING ubiquitin ligase". The Journal of Biological Chemistry. 277 (24): 22085–92. doi:10.1074/jbc.M201252200. PMID 11927591. 
  14. ^ Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM. "A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage". Current Biology. 10 (15): 886–95. doi:10.1016/s0960-9822(00)00610-2. PMID 10959836. 
  15. ^ a b Sengupta S, Robles AI, Linke SP, Sinogeeva NI, Zhang R, Pedeux R, Ward IM, Celeste A, Nussenzweig A, Chen J, Halazonetis TD, Harris CC (Sep 2004). "Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest". The Journal of Cell Biology. 166 (6): 801–13. doi:10.1083/jcb.200405128. PMC 2172115Freely accessible. PMID 15364958. 
  16. ^ Stewart GS, Wang B, Bignell CR, Taylor AM, Elledge SJ (Feb 2003). "MDC1 is a mediator of the mammalian DNA damage checkpoint". Nature. 421 (6926): 961–6. doi:10.1038/nature01446. PMID 12607005. 
  17. ^ Xu X, Stern DF (Oct 2003). "NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors". FASEB Journal. 17 (13): 1842–8. doi:10.1096/fj.03-0310com. PMID 14519663. 
  18. ^ Kobayashi J, Tauchi H, Sakamoto S, Nakamura A, Morishima K, Matsuura S, Kobayashi T, Tamai K, Tanimoto K, Komatsu K (Oct 2002). "NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain". Current Biology. 12 (21): 1846–51. doi:10.1016/s0960-9822(02)01259-9. PMID 12419185. 
  19. ^ Fernandez-Capetillo O, Chen HT, Celeste A, Ward I, Romanienko PJ, Morales JC, Naka K, Xia Z, Camerini-Otero RD, Motoyama N, Carpenter PB, Bonner WM, Chen J, Nussenzweig A (Dec 2002). "DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1". Nature Cell Biology. 4 (12): 993–7. doi:10.1038/ncb884. PMID 12447390. 
  20. ^ Ward IM, Minn K, Jorda KG, Chen J (May 2003). "Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX". The Journal of Biological Chemistry. 278 (22): 19579–82. doi:10.1074/jbc.C300117200. PMID 12697768. 

Further reading[edit]