Recruitment of ATM Protein to Double Strand DNA Irradiated with Ionizing Radiation

Abstract

The product of the ATM gene, which is mutated in ataxia telangiectasia, is a nuclear phosphoprotein, and it involves the activation of the p53 pathway after ionizing radiation. Here we show that the ATM protein is constitutively associated with double strand DNA and that the interaction increases when the DNA is exposed to ionizing radiation. The ATM protein also had affinity to restriction endonuclease PvuII-digested DNA, but not to UV-irradiated DNA nor X-irradiated single-stranded DNA. The immunoprecipitation experiment detected very weak association between ATM and DNA-PK proteins, and immunodepletion of DNA-PK showed little or no effect on the interaction of the ATM protein with damaged DNA, indicating that an interaction with DNA-PK might not be required for the recruitment of the ATM protein to damaged DNA. Furthermore, the association was also confirmed in xrs-5 and xrs-6e cells, which are Chinese hamster ovary mutant cell lines defective in Ku80 function. These results indicate that the ATM protein is recruited to the site of DNA damage and it recognizes double strand breaks by itself or through an association with other DNA-binding protein other than DNA-PK and Ku80 proteins. Ataxia telangiectasia (AT)1 is a human autosomal recessive disorder characterized by a variety of clinical symptoms. At the cellular level, AT cells exhibit hypersensitivity to ionizing radiation, radio-resistant DNA synthesis, and a high frequency of chromosome aberrations (reviewed in Refs. 1-3). Recently, the gene defective in AT cells has been cloned and designated ATM (4). Sequence analysis revealed that the ATM protein shows homology to a family related to phosphatidylinositol 3-kinase, those include phosphatidylinositol 3-kinase p110 subunit, FRAP, RAFT1, and DNA-PK proteins in mammalian cells (2,(5)(6)(7). Because p53 accumulation is diminished or severely delayed in AT cells irradiated with ionizing radiation, ATM protein has been suggested to be involved in p53 activation (8 -11). p53 is a nuclear protein whose phosphorylation is required for its stability and activity (reviewed in Refs. 12-15). Our study and others (16,17) have reported that ionizing radiation causes p53 protein accumulation, and recently phosphorylation of p53 at serine 15 has been shown to control p53 accumulation through inhibition of its interaction with MDM2 (18 -20). Furthermore, recent in vitro experiments have indicated that ATM phosphorylates p53 protein at serine 15 (21, 22). Although there has been a report indicating the phosphorylation of serine 15 in AT cells (23), it is highly possible that the activity of p53 protein is mediated by ATM kinase activity.Very little is known about the mechanism by which ATM protein is activated by DNA damage. Previous studies have shown that ATM protein levels do not change during cell cycle progression or after exposure to ionizing radiation (24 -26), and therefore, it has been hypothesized that ATM protein may not be an inducible effector, but may act as a transducer or, more likely, a sensor for DNA dama...

Contribution of the Atm Protein to Maintaining Cellular Homeostasis Evidenced by Continuous Activation of the AP-1 Pathway in Atm-deficient Brains

“…ATM is activated by DSBs induced by internal or external DNA damaging agents, such as IR, radiomimetic chemicals, and reactive oxygen species and is probably activated also by DSBs that occur normally during meotic and V(D)J recombination. ATM can bind to DNA in vitro (11,12), and a fraction of ATM binds tightly to the chromatin following the induction of DSBs, in accordance with the suggested role of ATM in the early steps of the DNA damage response.…”

Activation of p53 transcriptional activity requires ATM's kinase domain and multiple N-terminal serine residues of p53

“…The N-terminal of ATM can associate with both p53 (Khanna et al, 1998) and histone deacetylase in vitro (Kim et al, 1999), and both of these proteins co-precipitate with ATM in vivo (Khanna et al, 1998;Kim et al, 1999). ATM has also been shown to bind to dsDNA (Smith et al, 1999a;Suzuki et al, 1999), and this may involve sequences in the N-terminal of ATM (Smith et al, 1999b). Whether ATM binds directly to DNA or if this interaction is mediated through a speci®c DNA-binding sub-unit is not known.…”

hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

“…The PIKKs ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) function in parallel pathways to coordinate the cellular response to DNA damage. ATM plays a crucial role in regulating checkpoint activation in response to DNA doublestrand breaks as seen with ionizing radiation (Suzuki et al, 1999). While ATM can directly bind damaged DNA in vitro, it can also be recruited to sites of damage by a complex of Mre11, Rad50 and Nbs1 (MRN complex) (Smith et al, 1999;Abraham and Tibbetts, 2005;Lee and Paull, 2005).…”