Averbeck, N. B.

Becker, G.

Durante, M.

Jakob, B.

Leifke, A. L.

Ringel, O.

Taucher-Scholz, G.

GSI Repository

GSITemplate2007 The histone H2AX does not inhibit resection of DNA double strand breaks  induced by heavy ions N. B. Averbeck1, O. Ringel1, B. Jakob1,  A. L. Leifke1, G. Becker1, M. Durante1, 2,  and G. Taucher-Scholz1, 2  1GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany;  2Technical University Darmstadt, GermanyThe histone H2A variant H2AX plays an important role in the repair of DNA double strand breaks (DSBs) within eukaryotes. Very rapidly upon DSB induction this H2A variant becomes phosphorylated at Ser139 [1]; H2AX phosphorylated at this site is named γH2AX. It serves as a platform for several DSB repair factors [reviewed in 2] and further is thought to stabilize the loose ends of a DSB [3]. In addition, γH2AX controls the end processing of recombination activating gene (RAG) endonuclease in-duced DSBs within V(D)J recombination, which takes place in the G1 cell cycle phase of lymphocytes. Absence of γH2AX within this process allows CtIP-mediated DSB resection [4]. Hence, γH2AX prevents resection within the V(D)J recombination and it is conceivable that resec-tion of genotoxic caused DSBs is as well under the con-trol of γH2AX [4], especially in the cell cycle phase G1 when DSB resection would lead to genomic instability.   Studies here at GSI clearly demonstrated that CtIP-driven resection of heavy ion induced DSBs occurs not only in the late S and G2 cell cycle phase but in G1 as well [5] sustaing the notion that resection of ion induced DSBs in G1 is not inhibited by γH2AX. This is further supported by the observation that in ion irradiated conflu-ent human fibroblasts (AG1522D) (figure 1), where 98 % of cells were in the G1/G0 cell cycle phase, most DSBs are resected and the resection marker pRPA always colo-calizes with γH2AX at ion induced DSBs. There was not one cell where RPA was recruited to a break site where H2AX was not phosphorylated. This was observed in three independent experiments (2x 238U irradiation, LET: 15 000 keV/μm, fluence: 3 x 106 p./cm2; 1x 207Pb irradia-tion, LET 13500 keV/μm, fluence 3 x 106 p./cm2). Thus, γH2AX seems not to inhibit DSB resection of heavy ion induced lesions.        Figure 1: Resection of ion induced DSBs is never ob-served independently of γH2AX. Confluent human fi-broblasts were irradiated with 207Pb ions (LET:  13500 keV/μm; fluence: 3 x 106 p/cm2) and fixed 1 h after irradiation. Cells were immuno-stained for γH2AX (DSB marker) and pRPA (resection marker). DNA was visual-ized by DAPI staining.  In order to further characterize whether or not DSB re-section in G1 is controlled by H2AX and in particular by γH2AX we studied DSB resection in H2AX deficient cells. Murine H2AX -/- and wild-type cells were 12C ion irradiated (LET: 170 keV/μm; fluence: 3 x 106 p/cm2) and immuno-stained against RPA (resection marker) and 53BP1 (DSB marker) 1 h after irradiation. If γH2AX were inhibiting ion induced DSB resection in the G1 cell cycle phase one would expect that the H2AX deficient cells show an increase in resected DSBs. However, H2AX deficient and wild type cells both show about  20 % of resected DSBs. A different cell cycle distribution cannot be responsible for the obtained result as approxi-mately 50 % of both populations were in the G1 cell cycle phase (determined by flow cytometry). As H2AX defi-ciency did not influence the fraction of resected ion in-duced DSBs in G1 we conclude that γH2AX indeed does not inhibit resection of heavy ion induced DSBs in cells within the G1 cell cycle phase.   Taken together, from these observations we conclude that H2AX most likely has no function in controlling end resection of genotoxic-induced DSBs. Its influence on DSB resection might, thus, be unique to V(D)J recombi-nation. References [1] E.P. Rogakou et al., DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139, J Biol Chem, 273 (1998) 5858-5868. [2] J. Yuan et al., Focus on histone variant H2AX: to be or not to be, FEBS Lett, 584 (2010) 3717-3724. [3] B. Yin et al., Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination, J Exp Med, 206 (2009) 2625-2639. [4] B.A. Helmink et al., H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes, Nature, 469 (2011) 245-249. [5] N. Averbeck et al., DNA double strand break resection after heavy ion irradiation, in: K. Große (Ed.) GSI Scientific Report 2011, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, 2012.   Acknowledgements This work is partially funded by the BMBF grant 02NUK001A. GSI SCIENTIFIC REPORT 2012 HEALTH-03427

The histone H2AX does not inhibit resection of DNA double strand breaks induced by heavy ions

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The histone H2AX does not inhibit resection of DNA double strand breaks induced by heavy ions

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