4 Journal of Nucleic Acids Re-localization of DDR proteins (MRE11/NBS1/RAD50, MDC1, 53BP1,BRCA1) to nuclear foci where these proteins co-localize and interact with γ-H2AX DNA damage ATM activation Accumulation of DNA damage signaling and repair proteins at DSBs Chromatin remodeling to prevent dissociation of break ends and enhance DSB processing and repair eﬁciency DNA damage γ-H2AX foci Synapsis of DNA ends and rejoining Keeping the broken DNA ends together Functions of γ-H2AX in DNA damage response DNA damage repair ATM activation DNA damage Induction of DNA damage checkpoints Phosphorylation of cell cycle checkpoint- related factors by ATM (p53, Chk2) Signal ampliﬁcation and induction of DNA damage- sensitive cell cycle checkpoints Recruitment of cohesins to promote sister chromatid- dependent recombinational repair DNA damage γ-H2AX-dependent recruitment of cohesins Homology-directed repair of double-strand breaks Sister chromatid cohesion Figure 1: H2AX phosphorylation and its role in DNA damage response upon phosphorylation H2AX became critical player in DNA damage response. Activity of γH2AX could be summarized to following main points: accumulation of DNA damage signaling and repair proteins at DSBs The generation of DSBs triggers the relocalization of many DNA damage response (DDR) proteins such as MRE11/NBS1/RAD50, MDC1, 53BP1and BRCA1 to nuclear foci where these proteins colocalize and interact with γH2AX. Signal ampliﬁcation and induction of DNA damage-sensitive cell cycle checkpoints γH2AX contribute to the recruitment of ATM to DSB sites and activation of ATM-dependent cell cycle checkpoints. Chromatin remodeling to prevent dissociation of break ends and enhance DSB processing and repair eﬃciency The γH2AX foci help keeping the broken DNA ends together and make successful and faithful repair more likely. Recruitment of cohesins to the site of DNA damage to promote sister chromatid-dependent recombinational repair DNA cohesion induced by double-strand DNA break and mediated by γH2AX has an important function during repair of double-strand breaks following DNA replication by holding the damaged chromatid close to its undamaged sister template. facilitating DNA end rejoining, can be important not only in the repair of DSBs but also during V(D)J recombina- tion. According to hypothesis put forward by Bassing and colleagues, γH2AX behaves like an anchor that facilitates the assembly of multiple DNA-protein interactions involving 53BP1, MDC1/NFBD1, and MRN complexes. Formation of such complexes prevents dissociation of broken DNA ends and subsequent error-prone repair of DSBs (reviewed in [71]). Moreover, this speciﬁc “anchoring” function of H2AX at sites containing DSBs, would inhibit the irreversible disassociation of broken DNA ends and promote chromatin compaction to facilitate error-free repair, thereby suppress- ing inappropriate translocations of chromatin fragments. In this way, γH2AX-mediated mechanisms prevent DNA ends from drifting apart, inappropriate rejoining of chromatin fragments, resulting in genetic translocations and other abnormalities (Figure 1 and reviewed in [71]). 3.4. Recruitment of Cohesins to the Site of DNA Damage to Pro- mote Sister Chromatid-Dependent Recombinational Repair. γH2AX might be important for processes occurring further away from the break that are required for eﬃcient repair. One of such processes can be sister chromatid cohesion, that in addition to its essential role in chromosome segregation, is important for eﬃcient postreplicative double-strand DNA break repair [72]. Sister chromatid cohesion is established during S phase by cohesins. In the absence of cohesin complex, chromosomes cannot segregate properly. It was also shown that cohesins play an important role in DNA repair and recombination. Cells with mutated cohesins are oversensitive to irradiation and deﬁcient in postreplicative DNA repair [72, 73]. Moreover, cohesins have been shown to accumulate at laser-induced DNA damage during S and G2 phases in a Mre11/Rad50- dependent manner [74]. In parallel, it was shown that DNA cohesion induced by double- strand DNA breaks and mediated by γH2AX is not only important to maintain sister chromatids. It is also likely to function during repair of double-strand breaks following DNA replication by holding the damaged chromatid close to its undamaged sister template [64]. The use of genetic information present in an undamaged sister chromatid to mediate error-free recombination repair of a DSB would prevent gross chromosomal alterations, which are frequently