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 evolutionarily conserved signal for DNA damage.
Arun Gupta, Girdhar G Sharma, Charles SH Young, Manjula Agarwal, Edwin R Smith, Tanya T Paull, John C Lucchesi, Kum Kum Khanna, Thomas Ludwig, and Tej K Pandita. “Involvement of human MOF in ATM function.” Mol Cell Biol, 25, 12, Pp. 5292-305. Abstract
We have determined that hMOF, the human ortholog of the Drosophila MOF gene (males absent on the first), encoding a protein with histone acetyltransferase activity, interacts with the ATM (ataxia-telangiectasia-mutated) protein. Cellular exposure to ionizing radiation (IR) enhances hMOF-dependent acetylation of its target substrate, lysine 16 (K16) of histone H4 independently of ATM function. Blocking the IR-induced increase in acetylation of histone H4 at K16, either by the expression of a dominant negative mutant DeltahMOF or by RNA interference-mediated hMOF knockdown, resulted in decreased ATM autophosphorylation, ATM kinase activity, and the phosphorylation of downstream effectors of ATM and DNA repair while increasing cell killing. In addition, decreased hMOF activity was associated with loss of the cell cycle checkpoint response to DNA double-strand breaks. The overexpression of wild-type hMOF yielded the opposite results, i.e., a modest increase in cell survival and enhanced DNA repair after IR exposure. These results suggest that hMOF influences the function of ATM.
Double-strand breaks (DSBs) in chromosomal DNA elicit a rapid signaling response through the ATM protein kinase. Recent evidence suggests that the DNA repair complex containing Mre11, Rad50 and Nbs1 (MRN) is important for the activation of ATM by DSBs in cells. Our studies of the effects of MRN on ATM activity in vitro indicated that MRN stimulates ATM through multiple protein-protein contacts, and that this interaction increases the affinity of ATM for its substrates. Recently we isolated dimeric forms of ATM, which require MRN for activity but also require DNA, similar to the requirements for ATM activation and activity in vivo. Here we discuss the distinct characteristics of dimeric ATM, the role of MRN in recruiting ATM to DNA, the importance of DNA unwinding by MRN, and the role of autophosphorylation in ATM activation.
In Saccharomyces cerevisiae, the Mre11/Rad50/Xrs2 (MRX) complex plays important roles in both homologous and non-homologous pathways of DNA repair. In this study, we investigated the role of the MRX complex and its enzymatic functions in non-homologous repair of DNA ends containing incompatible end structures. Using a plasmid transformation assay, we found that mre11 and rad50 null strains are extremely deficient in joining of incompatible DNA ends. Expression of the nuclease-deficient Mre11 mutant H125N fully complemented the mre11 strain for joining of mismatched ends in the absence of homology, while a mutant of Rad50 deficient in ATP-dependent activities exhibited levels of end-joining similar to a rad50 deletion strain. Although the majority of non-homologous end-joining (NHEJ) products isolated did not contain microhomologies, introduction of an 8bp microhomology at mismatched ends resulted in microhomology-mediated joining in all of the products recovered, demonstrating that a microhomology exerts a dominant effect on processing events that occur during NHEJ. Nuclease-deficient Mre11p was less efficient in promoting microhomology-mediated end-joining in comparison to its ability to stimulate non-microhomology-mediated events, suggesting that Mre11p influences, but is not essential for, microhomology-mediated repair. When the linearized DNA was transformed in the presence of an intact homologous plasmid to facilitate gap repair, there was no decrease in NHEJ products obtained, suggesting that NHEJ and homologous repair do not compete for DNA ends in vivo. These results suggest that the MRX complex is essential for joining of incompatible ends by NHEJ, and the ATP-dependent activities of Rad50 are critical for this process.
At least three DNA polymerases participate in nonhomologous end joining in mammalian cells: pol mu, pol kappa, and TdT. A study in this issue of Molecular Cell (Nick McElhinny et al., 2005) clarifies the role of pol mu in end joining at the kappa light chain locus and also provides a biochemical explanation for the unique polymerization functions of pol mu on DNA ends.
