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Recruitment of the Rad BP mediator
Recruitment of the Rad9/53BP1 mediator to AMD-070 involves multiple pathways (Fig. 2). In unperturbed conditions, Rad9 is already bound to chromatin via interaction between its Tudor domain and methylated histone H3 at lysine 79 [82], [83], [84], [85]. This constitutive Rad9 recruitment to chromatin is thought to facilitate the efficiency of the Rad9-dependent response to DNA damage, which requires additional histone modifications. In fact, Rad9 binding to the sites of damage is further strengthened by the interaction between its BRCT domain with histone H2A that has been phosphorylated at serine 129 (γH2A) [82], [84], [86], [87], [88]. Similarly, 53BP1 binding to DSBs is facilitated by phosphorylation of the histone variant H2AX at serine 139 [89], [90].
Activation of ATM/Tel1
It is known that ATM is activated primarily by DSBs, but the specific signals that activate ATM following DSB induction are still unclear. It has been suggested that the initial trigger of ATM activation might be a modification in chromatin structure surrounding the DSB rather than direct contact of ATM with broken DNA [91]. Full activation of human ATM is dependent on autophosphorylation on Ser1981 and interaction with the MRN complex at the DSB sites. ATM exists as an inactive dimer in unperturbed cells, but it undergoes intermolecular autophosphorylation on Ser1981 after DSB formation or treatment with agents that alter chromatin structure, resulting in dissociation of the dimer into active monomers [91]. Besides Ser1981, other autophosphorylation sites (Ser367, Ser1893 and Ser2996) play a role in the ATM activation process [92], [93]. Although the functional significance of ATM autophosphorylation is still unclear, it has been recently shown to be required for the stabilization of activated ATM at DSB sites, albeit not for the initial ATM recruitment [94], [95].
ATM/Tel1 activation requires also the MRN/MRX complex. Mre11 contains a C-terminal DNA binding domain as well as a phosphoesterase domain that provides ssDNA endonuclease and 3′–5′ dsDNA exonuclease activities [13], [14], [15]. Both Rad50 and Xrs2/Nbs1 enhance the nuclease activity of Mre11 in vitro, and Rad50 has in vitro ATP-binding and hydrolysis activities that are critical for MRN/MRX function [96]. Notably, cells defective in any component of the MRN/MRX complex are defective in ATM/Tel1 activation, indicating that this complex is crucial for ATM/Tel1 function. Which is the exact molecular mechanism of ATM/Tel1 activation by MRN/MRX remains to be elucidated, although it is clear that ATM/Tel1 is recruited to sites of DNA DSBs through its interaction with the C-terminal domain of Nbs1/Xrs2 [97], [98], [99], [100]. In mammals, direct tethering of a large number of Mre11, Nbs1 or ATM molecules to a specific chromosome locus activates ATM even in the absence of DSBs [101], suggesting that one of the functions of the ATM-MRN interaction is to accumulate ATM/Tel1 at the damage sites. Interestingly, the action of Mre11 at the DSB ends has been reported to produce small DNA fragments that can stimulate ATM activity [102], raising the possibility that the MRN/MRX complex has other functions besides the recruitment of ATM/Tel1 at the DSBs. Furthermore, Tel1 kinase activity is stimulated by MRX binding to DNA-protein complexes at DSBs [103], suggesting that the MRX complex might control Tel1 catalytic activity by monitoring protein binding at DNA ends.
Activation of ATR/Mec1
Although ATR is primarily activated by replication stresses, its activation can be promoted also by DNA DSBs. However, in mammals ATR activation is slower than ATM activation and occurs predominantly in the S/G2 phase of the cell cycle [104]. In both mammals and yeast, recruitment of ATR/Mec1 at the DSB sites requires the presence of RPA-coated ssDNA 3′ overhangs [105], which are generated by nuclease-mediated DSB resection (Fig. 2). Recognition of RPA-coated ssDNA by ATR/Mec1 depends on an ATR/Mec1 interacting protein, called ATRIP in mammals and Ddc2 in S. cerevisiae. Biochemical studies indicate that ATRIP binds the RPA complex directly and this interaction involves an ATRIP acidic α-helix that binds to the basic cleft of the N-terminal oligonucleotide/oligosaccharide binding (OB)-fold domain of the large RPA1 subunit [106]. Loss of ATRIP/Ddc2 causes the same phenotypes as loss of ATR/Mec1 in both mammals and yeast, indicating that ATRIP/Ddc2 is required for all known ATR/Mec1 functions [107], [108].