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  • How does the ATM to ATR switch occur at DSBs

    2022-11-18

    How does the ATM-to-ATR switch occur at DSBs? The progressive attenuation of ATM activation could be attributed to the loss of DNA structures that activate ATM, or to the generation of DNA structures that interfere with ATM activation. Our finding that SSOs do not directly affect the binding of purified MRN to dsDNA, and that ssDNA interferes with ATM activation in extracts both in cis and in trans, supports the latter possibility. Both the recruitment of ATR-ATRIP and the interference with ATM activation are dependent on the length of SSOs. As DSBs are progressively resected by nucleases, SSOs are gradually lengthened, simultaneously enhancing the abilities of SSOs to interfere with ATM activation and to promote ATR activation (Figure 7F). We show that SSOs attenuate the binding of MRN to dsDNA in extracts (Figure 3E) but facilitate the recruitment of RPA and ATRIP (Figure 5B). These results suggest that SSOs promote a swap of DNA-damage sensors at DSBs, revealing the underlying mechanism for the ATM-to-ATR switch. Interestingly, recent studies suggested that the resection of DSBs by nucleases is also a biphasic process: it DCPIB is initiated by the MRN-CtIP complex and then extended by the Exo1- or BLM-dependent mechanisms (Gravel et al., 2008, Mimitou and Symington, 2008, Zhu et al., 2008). It is plausible that the release of MRN from resected DNA ends may link the nuclease switch with the ATM-to-ATR switch during DSB resection. We propose that the ATM-to-ATR switch driven by DSB resection is the key mechanism through which the functions of ATM and ATR are coordinated and integrated. The ATM-to-ATR switch is distinct from, but complementary with, the sequential activation of ATM and ATR reported previously (Jazayeri et al., 2006). Together, these two regulatory mechanisms ensure that ATM launches DSB response, whereas ATR plays a primary role in the second phase of this dynamic process. The associations of ATM and ATR with DSBs are not only temporally but also spatially distinct (Bekker-Jensen et al., 2006) and are differentially influenced by the cell cycle (Huertas et al., 2008, Ira et al., 2004, Jazayeri et al., 2006, Zierhut and Diffley, 2008). Moreover, the levels of ATM and ATR activation can be fine-tuned by DSB resection (Mantiero et al., 2007, Vaze et al., 2002, Zierhut and Diffley, 2008). The ATM-to-ATR switch may enable the two kinases to target distinct sets of downstream effectors in a regulated and coordinated manner. For instance, this switch may couple ATM and ATR with distinct events during DSB repair. The ATM-to-ATR switch driven by DSB resection may provide a unifying mechanism that brings together the temporal, spatial, and quantitative regulations of ATM and ATR, thus orchestrating the collective checkpoint response in human cells. This study has set the stage for future investigations to reveal how the ATM-to-ATR switch conducts the specific functions of ATM and ATR during the biphasic DSB response.
    Experimental Procedures
    Acknowledgments
    The induction of apoptosis is crucial for eliminating cells with damaged DNA or deregulated proliferation. Tumor suppressors p53 and ARF are the most frequently abrogated genes found in human cancers, suggesting that they are the central components of the cellular defense against cell transformation. Although p53 and ARF prevent tumor formation both in concert and also separately, disruption of both p53 and ARF in same malignancies is detected relatively rarely , , . In normal cells the level of p53 protein is low due to its short half-life . The main regulator of p53 protein levels is Mdm2 protein, which inhibits p53 transactivator properties and marks p53 for proteasomal degradation through its ubiquitinylation , . p53 is activated and stabilized in response to cellular stress caused by hyperproliferative signals, hypoxia, and DNA damage by irradiation or chemotherapeutic drugs. Activated p53 induces or represses the transcription of a variety of genes, leading to either cell cycle arrest or apoptosis , .