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The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. We show that the Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation was not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN was essential for ATM stimulation, which is consistent with the central role of single-stranded DNA as an evolutionary conserved signal for DNA damage.
In mammalian cells, DNA double-strand breaks trigger activation of the ataxia-telangiectasia mutated (ATM) protein kinase, which phosphorylates downstream targets that initiate cell-cycle arrest, DNA repair, or apoptosis. Several of these targets, including p53, Chk2, Brca1, and H2AX, function as tumor suppressors in vivo, and the phosphorylation of these factors is critical for their function after DNA damage.
The Nbs1 (nibrin) protein is also a substrate for ATM, and abrogation of Nbs1 phosphorylation inhibits checkpoint signaling during the S phase (chromosome replication) of the cell cycle (1). Nbs1 is part of the Mre11-Rad50-Nbs1 (MRN) complex, which is essential for DNA double-strand-break repair and genomic stability. Cells from patients with Nijmegen breakage syndrome (NBS) or ataxia telangiectasia-like disorder (ATLD) express mutant forms of the Nbs1 or Mre11 protein, respectively, and exhibit decreased levels of ATM substrate phosphorylation, particularly on Chk2 (2-5) and Smc1 (6, 7), despite the presence of wild-type ATM. Thus, the MRN complex may not only be a downstream effector of ATM but also may function in activating ATM to initiate phosphorylation of cellular substrates.
MRN stimulated ATM activity in vitro toward p53, Chk2, and histone H2AX in a kinase assay with purified recombinant components (8). MRN and ATM associated through multiple protein-protein interactions, and MRN contributed to ATM kinase activity by increasing the affinity of ATM for its substrates. In this in vitro assay, however, there was no effect of DNA on ATM, either with or without MRN (Fig. 1A).
ATM exists in vivo as an inactive multimer that dissociates into active monomers after DNA damage or other forms...