br Results br Discussion Both ATM and

Results
Discussion Both ATM and ATR are key regulators of the cellular response to DSBs, yet how exactly they function in concert is not well understood. Recent studies revealed that ATM is required for the resection of DSBs (Jazayeri et al., 2006, Myers and Cortez, 2006), a process necessary for ATR activation as well as homology-directed DNA repair. While these studies established a critical function of ATM in initiating DSB response, they have not resolved how ATM and ATR function during the dynamic process of DSB resection, a crucial INF39 for both damage signaling and DNA repair. A major obstacle to understanding the coordination of ATM and ATR is that the DNA structural elements regulating ATM activation have not been clearly defined. In this study, we developed an extract-based in vitro assay in which both ATM and ATR can be activated by dsDNA in a DNA structure-regulated manner. Using this assay, we systematically characterized the DNA structural determinants for ATM activation as well as the orchestration of ATM and ATR at DSBs. The results of this study addressed two important issues with regard to the activation of ATM at resected DSBs: Can ATM be activated by resected DSBs? And if so, is ATM activated by the ends of SSOs or the junctions of single-/double-stranded DNA? Our results clearly demonstrated that ATM can be activated in the presence of short SSOs and, furthermore, that ATM activation relies on the junctions of single-/double-stranded DNA, but not the ends of SSOs (Figure 4). Given that the activation of ATM by dsDNA requires Nbs1, it is plausible that the MRN complex directly recognizes the junctions of single-/double-stranded DNA when SSOs are present. We also show that paired DNA ends are important for this recognition step (Figure 2E). The MRN complex bound to DNA ends may directly activate ATM and/or initiate the nucleation of ATM at DSBs that leads to its activation. The recognition of DNA ends by MRN may be a prerequisite for its DNA unwinding, tethering, or nuclease activities implicated in ATM activation (Costanzo et al., 2004, Jazayeri et al., 2008, Lee and Paull, 2005, Uziel et al., 2003). Our results also reveal that the activation of ATM is coordinately regulated by three distinct DNA structural elements of DSBs: (1) DNA ends, (2) dsDNA, and (3) ssDNA. Notably, ATM activation is oppositely regulated by the two DNA structures accompanying DNA ends: it is enhanced by flanking dsDNA, but hindered by SSOs. Both of these regulatory mechanisms operate in a length-dependent manner and may function in concert to quantitatively regulate ATM activation at DNA ends. Interestingly, ATM is involved in the resection of DNA breaks (Jazayeri et al., 2006), suggesting that the activation of ATM elicits an inhibitory feedback loop through SSO formation. While these results present a clear picture of how ATM activation is regulated by the structures of DNA at DSBs, how the DNA- and chromatin-mediated regulatory mechanisms are integrated during ATM activation remains to be determined (Bakkenist and Kastan, 2003, Lou et al., 2006, You et al., 2007). The in vitro ATM activation assay described here may provide the basis for future biochemical studies to dissect the concerted action of DNA and chromatin in ATM activation. The involvement of dsDNA/ssDNA junctions in ATM activation reveals an unexpected similarity between the DNA structural specificities of ATM and ATR, suggesting that the choice between activating ATM or ATR at a resected DSB is made by another DNA structure. We have previously shown that ssDNA coated by RPA is the key structure that enables the ATR-ATRIP kinase complex to recognize DSBs (Zou and Elledge, 2003). Our finding that SSOs interfere with ATM activation immediately raised the possibility that ATR activation is coupled with loss of ATM activation through ssDNA. Consistent with this model, RPA gradually accumulates at DSBs (Figure 6C), whereas Nbs1 associates with DSBs rapidly and transiently when it is unable to retain on the flanking chromatin (Celeste et al., 2003). The activation of yeast Tel1(ATM) is attenuated as DSBs are progressively resected (Mantiero et al., 2007). We find that Chk2, a specific substrate of ATM, is rapidly and transiently phosphorylated after IR treatment. Furthermore, the ATR-dependent Chk1 phosphorylation lags behind Chk2 phosphorylation, and Chk1 phosphorylation increases as Chk2 phosphorylation declines (Figure 6). Collectively, these results provide compelling evidence that an ATM-to-ATR switch indeed occurs in human cells in response to DSBs.