Journal of Nucleic Acids MDC1, 53BP1, and BRCA1 to nuclear foci where these pro- teins colocalize and interact with γH2AX [36–46]. Presum- ably, γH2AX foci speciﬁcally attract repair factors, leading to higher concentration of repair proteins surrounding a DSB site [47, 48]. Speciﬁc recognition of γH2AX by these repair factors requires the presence of protein domains, which bind to the phosphorylated carboxy terminus of γH2AX. So far, two domains, which are frequently found in proteins involved in DDR, have been described to specif- ically recognize phosphorylated amino acid residues. The forkhead-associated (FHA) domain recognizes phosphory- lated threonine residues in a speciﬁc aminoacid sequence context [49]. In addition, two consecutive BRCT domains (BRCA1 C-terminal domain) can create a structural element with phospho–peptide binding capacity [50–63]. It was shown that the BRCT repeats of mediator of DNA dam- age checkpoint protein 1 (MDC1) build the predominant recognition module of γH2AX [39, 43, 54, 55]. Interaction between MDC1 and γH2AX can be recognized as the ﬁrst step in which the site of the DSB is prepared for DNA damage signaling and repair. This is because there is experimental evidence that MDC1 also directly interacts in a highly dynamic manner with NBS1 [43, 56], which together with other proteins of the MRN complex is required for the activation of ATM [57, 58]. This interaction is mediated through phosphorylation of MDC1 by CK2 that, in turn, promotes phosphorylation-dependent interactions with NBS1, through its closely opposed FHA domain and two BRCA repeats [59]. In this way, a positive feed-back loop is generated that extends H2AX phosphorylation to large DNA regions (millions of base pairs). Several lines of evidence suggest the critical role of H2AX phosphorylation at DSB sites for nuclear foci formation and induction of DSB repair. (i) H2AX-knockout cells manifested impaired recruit- ment of NBS1, 53BP1, and BRCA1 to irradiation- induced foci [45]. (ii) Both H2AX+/− and H2AX−/− mouse thymocytes show an increase in chromosomal aberrations [60– 62]. (iii) Mouse embryonic stem (ES) cells deﬁcient in H2AX phosphorylation have alterations in eﬃciency of DNA repair by NHEJ or HR [60–64]. As a result of these defects in DNA damage repair, such cells have increased sensitivity to DNA damage [60–65]. (iv) H2AX knock-out mice show male-speciﬁc infertility and reduced levels of secondary immunoglobulin isotypes, suggesting defects in class switch recom- bination (CSR) [45]. It was shown that eﬃcient resolution of DSBs induced during CSR in lympho- cytes requires H2AX [61, 63], and its absence is associated with chromosome abnormalities involving the immunoglobulin locus [61]. All these facts suggest that γH2AX might serve as a docking site for DNA damage/repair proteins and functions to promote DSB repair and genome stability ([60–68] and Figure 1). 3 3.2. Signal Ampliﬁcation and Induction of DNA Damage- Sensitive Cell Cycle Checkpoints. One of most important kinases-activating cell cycle checkpoints following DNA damage is ATM. Mutations in the ATM gene results in the genomic instability and cancer predisposition syndrome Ataxia-Telangiectasia (AT) syndrome. In response to DSBs, ATM phosphorylates many cell cycle checkpoint-related factors such as p53, Chk2, SMC1 and NBS1 [69]. In the absence of DNA damage, ATM forms inactive homodimers, but chromatin remodeling following generation of DSBs leads to autophosphorylation of ATM at serine 1981 and dimer dissociation [18–21]. Next, phosphorylated ATM is recruited to DSB sites by its interaction with NBS1 and this recruitment is critical for phosphorylation of checkpoint proteins by ATM [57]. Mutations in the NBS1 gene result in Nijmegen breakage syndrome (NBS). Cells from both NBS and AT patients show similar phenotypes such as radio-resistant DNA synthesis, radiation hypersensitivity and genome instability [38]. As it was previously described, NBS1 forms a complex with hMRE11 and hRAD50. Since such a complex is involved in DNA double-strand break repair by homologous recombination (HR), one of the reasons of the genomic instability in NBS patients may be the defect in HR. Moreover, it was shown that this function of NBS1 requires DNA damage-induced focus formation of the NBS1/hMRE11/hRAD50 complex through direct interaction of NBS1 with γH2AX at DNA damage sites [37, 70]. As mentioned above, the recruitment of ATM to DSBs is dependent on NBS1 and NBS1 forms the complex with both ATM and γH2AX. Thus, both γH2AX and NBS1 contribute to the recruitment of ATM to DSB sites and its activation of cell cycle checkpoints. Molecular mechanism involved in ATM recruitment to DSBs was recently investigated in detail by Kobayashi and colleagues [38]. These authors provide a line of evidence that γH2AX plays an important role in the recruitment of ATM to DSB sites and the subsequent acti- vation of ATM-related cellular response. Moreover, H2AX is a component of the complex containing ATM at DSBs and is important for activation of the ATM kinase. Importantly, H2AX-knockout cells displayed a defect in DSB-induced cell cycle checkpoint response. These results provide strong evidence that H2AX is one of essential components of the active ATM complex and participates in the activation of ATM-dependent cell cycle checkpoints. Moreover, Kobayashi and colleagues showed that ATM forms a complex with γH2AX via NBS1 or MDC1 and this might be crucial for the recruitment of ATM to DSB sites and induction of ATM- dependent checkpoints [38]. 3.3. γ-H2AX and Chromatin Remodeling to Prevent Dissoci- ation of Break Ends and Enhance DSB Processing and Repair Eﬃciency. During last few years several groups suggested that γH2AX has an important function as a docking site for protein complexes that bind to broken DNA ends and promote chromatin remodeling to keep broken chromo- somal DNA ends together [60, 63, 71]. The critical role of γH2AX in chromatin reorganization following DNA damage and preventing the separation of DNA ends, thus