The interaction of ATR ATRIP with RPA coated

The interaction of ATR-ATRIP with RPA-coated ssDNA is sufficient for ATR-ATRIP recruitment to DNA lesions, but it is not sufficient to fully activate ATR. In response to DNA damage, the heterotrimeric ring-shaped complex 9-1-1 (RAD9-RAD1-HUS1 in humans; Ddc1-Rad17-Mec3 in S. cerevisiae) is loaded at the junctions between ssDNA and dsDNA by the RFC-like clamp loader RAD17-RFC2-5 in humans or Rad24-Rfc2-5 in S. cerevisiae. In NVP-BHG712 synthesis yeast, co-localisation of Mec1-Ddc2 and 9-1-1 at damage sites directly stimulates Mec1 kinase activity [109], [110]. Evidence demonstrating that the 9-1-1 clamp can similarly stimulate ATR/Rad3 kinase activity in vertebrates and S. pombe is lacking. Rather, the 9-1-1 complex has been proposed to stimulate ATR kinase activity by recruiting TopBP1 via an interaction between phosphorylated RAD9 and TopBP1 [111], [112], [113], [114]. This interaction facilitates the association of TopBP1 with ATRIP, which in turn stimulates ATR kinase activity. TopBP1-mediated activation of ATR is conserved in S. cerevisiae, where the Ddc1 subunit of the 9-1-1 complex recruits the TopBP1 ortholog Dpb11 to DNA damage sites [115], [116], [117], [118]. Phosphorylation of Ddc1 by Mec1 is critical for Dpb11 function in checkpoint signaling, suggesting that RPA-recruited Mec1-Ddc2 may have sufficient activity to phosphorylate Ddc1 before Ddc1-Dbp11 interaction takes place [118]. Interestingly, some data in both yeast and mammals suggest that the MRN/MRX complex is involved in ATR/Mec1 activation [119], [120]. Because MRN/MRX has a role in DSB resection, it has been proposed that MRN/MRX effects on ATR/Mec1 may be due to DSB processing. However, by using defined ATR-activating DNA structures in Xenopus egg extracts, MRN has been shown to recognize the ss/dsDNA junctions and to recruit TopBP1 to DNA [121]. Furthermore, Nbs1 is important for ATR-dependent phosphorylation of RPA. This function does not require 9-1-1 and can be separated from ATM activation and DSB resection [122]. Taken together, these findings lead to a working model where the ss/dsDNA junctions at the DSBs have two roles in ATR activation: activating TopBP1 by facilitating its interaction with the 9-1-1 complex, and recruiting TopBP1 by promoting its association to the MRN complex.
Interplays between ATM/Tel1 and ATR/Mec1 signaling A challenging question is how ATM and ATR actions are coordinated at DSBs. Interestingly, while activation of both ATM and ATR depends on the ss/dsDNA junctions, they are oppositely regulated by the lengthening of single-stranded overhangs [123]. Blunt double-strand ends, as well as ends with short single-stranded tails, are the preferred substrates for ATM activation. As the single-stranded tail increases in length, it simultaneously potentiates ATR activation and attenuates ATM activation [123]. A similar mechanism has been proposed for budding yeast Tel1, whose signaling activity is disrupted when the DSB ends are subjected to 5′–3′ exonucleolytic degradation [124]. These data suggest that the nature of the DSB ends is important to dictate the engagement of ATM/Tel1 or ATR/Mec1. In both humans and yeast, ATM/Tel1 activation promotes the accumulation of ssDNA at DSB ends and therefore is critical for the subsequent activation of ATR/Mec1 [104], [120], [123], [124], [125]. As generation of ssDNA ultimately leads to ATM inactivation, this mechanism ensures an efficient switch from ATM/Tel1 to ATR/Mec1. How ATM/Tel1 promotes DSB resection is unknown. One possibility is that ATM/Tel1 influences the function of the MRN/MRX complex in a positive feedback loop. Consistent with this hypothesis, Tel1 was recently shown to regulate the generation of ssDNA at telomeres by promoting MRX function [126]. Although MRN/MRX is required for ATM/Tel1 recruitment in all the species analyzed so far, different organisms apparently exhibit differences in the roles of ATM and ATR orthologs in checkpoint activation. For example, Tel1-deficient S. cerevisiae cells do not show obvious hypersensitivity to DNA damaging agents and are not defective in checkpoint activation in response to a single DSB [124]. This might be due to differences between ATM and Tel1 in their intrinsic kinase activity and/or in their ability to interact with specific targets and/or DNA–protein complexes at DNA ends. On the other hand, the ability of ATM/Tel1 to signal at a DSB is attenuated when the DSB ends are resected [124]. This finding suggests that the apparent minor role of Tel1 in DSB signaling may be explained by the ability of yeast cells to rapidly convert the DSB ends into ssDNA substrates that preferentially stimulate Mec1 kinase activity. Thus, although Tel1 contribution to the checkpoint can be masked by the prevailing activity of Mec1, the mechanism governing ATM- and ATR-dependent checkpoint activation in humans operates also in S. cerevisiae.