The ATM protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981 that is contained within the previously described 'FAT' domain. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell. Activation of the ATM kinase seems to be an initiating event in cellular responses to irradiation, and our data indicate that ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin.
The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. Here, we show that NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and that this recruitment is required for the phosphorylation of SMC1 by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation.[Keywords: ATM; NBS1; SMC1; BRCA1; DNA damage] Supplemental material is available at http://www.genesdev.org.
The phosphoinositide 3-kinase related kinases (PIKKs) mediate responses to diverse stresses, including DNA double-strand breaks (DSBs), abnormal replication fork progression, the recognition of premature termination codons in mRNAs, and inadequate nutrient availability. Rigorous control of these kinases limits cellular damage and promotes cell viability in the presence of stress. Control mechanisms include the localization of PIKKs into multiprotein complexes at the sites of damage and mediation of protein-protein contacts such that substrates are allowed access to the PIKK catalytic domains.
The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.
SummaryMitochondrial fission and fusion cycles are integrated with cell cycle progression. In this paper, we demonstrate that the inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by a hyperfused mitochondrial structure and unscheduled expression of cyclin E in the G2 phase. This persistent replication stress then induces an ATM-dependent activation of the G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and reactive oxygen species (ROS) generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates the DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.
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