2 and tangles from the genetic material by creating transient breaks in DNA double-helix [11, 12]. There are two classes of topoisomerases: type I enzymes introduce single-strand breaks in DNA and type II ones introduce double-strand breaks [11–13]. Type I topoisomerases modulate DNA under- and overwinding, but cannot remove knots and tangles from duplex DNA, while type II topoisomerases modulate DNA supercoiling and also remove DNA knots and tangles [14]. Since a single unrepaired DSB has potentially lethal consequences, type II topoisomerases, being necessary for cell survival, also have the capacity to fragment the genome [14]. When a nucleic acid tracking system, such as a replication or transcription complex, attempts to traverse the cleavable complex between topoisomerase and DNA, it may convert this transient enzyme-DNA intermediate to a permanent DSB in the genetic material, leading to DNA aberrations [15]. Inhibitors of DNA topoisomerase I (camptothecin and topotecan) and II (etoposide, doxorubicin, and mitox- antrone) belong to the most eﬀective antitumor drugs. Their mode of action involves stabilization of cleavable complexes between topoisomerase and DNA. It leads to collisions of the progressing DNA replication forks or RNA polymerase with the drug-stabilized topoisomerase-DNA complexes and conversion them into DSBs, which trigger apoptosis [16, 17]. In eukaryotic cells, the eﬃcient repair of DSB is essential for survival. Two major pathways have evolved to deal with these lesions, homologous recombination (HR), and non homologous end-joining (NHEJ). The mechanism of HR is based on using the genetic information from a corresponding undamaged region present on the second DNA molecule or homologous chromosomes and therefore is active mainly during S and G2 phases of cell cycle. NHEJ is based on a direct ligation of the two ends of damaged DNA molecules and repairs DSBs mainly in G1 phase [2]. 2. H2AX Phosphorylation It was reported several years ago, that in mammalian cells the phosphorylation of the subtype of histone H2A, called H2AX, in the position of Ser139 occurs in response to DSB formation. The phosphorylated form of H2AX is called γH2AX [18, 19]. Since then, many researchers focused on the explanation of mechanisms which induce phosphorylation of H2AX and its role in DNA damage signaling and repair. Histone H2AX is a substrate of several phosphoinositide 3-kinase-related protein kinases (PIKKs), such as ATM (ataxia teleangiectasia mutated), ATR (ATM and Rad3- related), or DNA-dependent protein kinase (DNA-PK). ATM kinase is considered as a major physiological mediator of H2AX phosphorylation in response to DSB formation [20, 21]. ATM is activated by its autophosphorylation at Ser1981 position, which leads to dissociation of the inactive ATM dimers into single protein molecules with increased kinase activity [18–21]. A tri-protein complex called MRN complex (MRE11-RAD50-NBS1) recognizes DNA damage, recruits ATM to the site of damage and also functions in targeting ATM to initiate phosphorylation of the respective Journal of Nucleic Acids substrates [22–24]. It is also reported that ATM activation requires prior ATM acetylation, mediated by Tip60 histone acetyltransferase [24, 25]. Apart from H2AX, the target substrates phosphorylated by ATM are BRCA1, 53BP1, and MDC1 as well as checkpoint proteins, Chk1 and Chk2. These processes are aimed to stop the progression of the cell cycle and to activate proteins responsible for DNA repair, as is described in details below. H2AX can also be phosphorylated by ATR and DNA- dependent protein kinases. ATR phosphorylates H2AX in response to single-stranded DNA breaks and during replica- tion stress, such as replication fork arrest [26, 27]. DNA-PK mediates phosphorylation of H2AX in cells under hypertonic conditions and during apoptotic DNA fragmentation [28, 29]. However, DNA damage caused by ionizing radiation leads to phosphorylation of H2AX that is mediated by all PIKK kinases, ATM, ATR, and DNA-PK [30]. Recently, it was reported that some other events occur before H2AX phosphorylation in mammalian cells. Ayoub et al. [31] observed that DNA breaks mobilize heterochromatin protein 1β (HP1-β), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). In response to DNA damage, HP1-β was rapidly phosphorylated at threonine 51 (Thr51) by casein kinase 2 (CK2). This phosphorylation leads to releasing HP1-β from chromatin by disrupting hy- drogen bonds that fold its chromodomain around H3K9me, resulting in its transient mobilization from chromatin. Although it is still not known what signals CK2 to phospho- rylate HP1, the phosphorylation and mobilization of HP1 seems to be important for H2AX phosphorylation [31]. Based on the results of Ayoub et al. [31] and Goodarzi et al. [32, 33], it is expected that the loss of HP1 is beneﬁcial to repair in heterochromatin. However, another recent study by Luijsterburg et al. [34], that also addressed the role of HP1 proteins in DNA damage response, suggested an apparently active and positive role for HP1 in DNA repair. These authors demonstrate that HP1 proteins accumulate de novo at sites of DNA damage, and this recruitment was independent of binding to H3K9me. It cannot be excluded that there is a partial HP1 dissociation from H3K9me at damaged sites, however the accumulation of HP1 reﬂects H3K9me-independent binding to damage sites rather than the rebinding of HP1 to H3K9me sites [34, 35]. Despite these controversies, the ﬁndings presented above illustrate the important role of HP1 proteins in DNA damage and open up a new direction of research to characterize the role of HP1 in H2AX phosphorylation and DNA damage response. 3. γH2AX and Its Role in DNA Damage Response 3.1. Accumulation of DNA Damage Signaling and Repair Pro- teins at DSBs. Unrepaired DSBs induce genome instability and promote tumorigenesis. Thus, cells have mechanisms responsible for recognition of DNA damage and activation of cell cycle checkpoints leading to DNA repair. The generation of DSBs triggers the relocalization of many DNA damage response (DDR) proteins such as MRE11/NBS1/RAD50,