The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity.Ataxia telangiectasia (A-T) is an inherited disease characterized by immune deficiencies, neurodegeneration, susceptibility to cancer, and sensitivity to ionizing radiation (28). The A-T gene product, the ATM protein kinase, is activated in response to DNA double-strand breaks (DSBs) (24, 35) and phosphorylates multiple DNA damage response proteins, including Nbs1, p53, chk2, and SMC1 (reviewed in reference 24). The phosphorylation of these proteins by ATM is essential for correct activation of cell cycle checkpoints and for the initiation of DNA repair. Consequently, cells lacking functional ATM protein exhibit defects in DNA repair and a loss of cell cycle checkpoints (24,28,35), resulting in increased sensitivity to ionizing radiation.A central question in studying the ATM protein has been to determine the mechanism by which ATM's kinase activity is activated by DNA damage. Activation of ATM's kinase activity is associated with increased autophosphorylation of ATM at multiple sites (23), including serine 1981 (2). This autophosphorylation of ATM is proposed to initiate a dimer-monomer transition and the release of active ATM monomers (2), although the exact contribution of ATM autophosphorylation to ATM activation is still under debate (31). Several additional molecular events, including the Mre11-Rad50-Nbs1 (MRN) DNA binding complex (8,11,14,41) and changes in chromatin structure (2, 4), also contribute to activation of ATM's kinase activity. Mutations in the individual protein components of the MRN complex can reduce or abolish the activation of ATM's kinase activity by DNA damage (8,11,14,41). The recent characterization of a conserved C-terminal domain in Nbs1 which is essential for recruitment of ATM to the MRN complex and for the efficient activatio...

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Cell cycle arrest is critical for muscle differentiation, and the two processes are closely coordinated but temporally separable. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that have been shown to be required for muscle differentiation in cell culture and have also been reported to be required for Rb-mediated cell cycle arrest. We therefore looked more closely at how SWI/SNF enzymes affect the events that occur during MyoD-induced myogenesis, namely, cell cycle regulation and muscle-specific gene expression, in cells that inducibly express dominant negative versions of Brahma (BRM) and Brahma-related gene 1 (BRG1), the ATPase subunits of two distinct SWI/SNF complexes. Although dominant negative BRM and BRG1 inhibited expression of every muscle-specific regulator and structural gene assayed, there was no effect on MyoD-induced activation of cell cycle regulatory proteins, and thus, cells arrested normally. In particular, in the presence or absence of dominant negative BRM or BRG1, MyoD was able to activate expression of p21, cyclin D3, and Rb, all of which are critical for cell cycle withdrawal in the G 1 /G 0 phase of the cell cycle. These findings suggest that at least one basis for the distinct mechanisms that regulate cessation of cell proliferation and musclespecific gene expression during muscle differentiation is that SWI/SNF-mediated chromatin-remodeling enzymes are required only for the latter.

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The SWI/SNF enzymes belong to a family of ATP-dependent chromatin remodeling enzymes that have been functionally implicated in gene regulation, development, differentiation and oncogenesis. BRG1, the catalytic core subunit of some of the SWI/SNF enzymes, can interact with known tumor suppressor proteins and can act as a tumor suppressor itself. We report that cells that inducibly express ATPase-deficient versions of BRG1 increase in cell volume, area of attachment and nuclear size upon expression of the mutant BRG1 protein. Examination of focal adhesions reveals qualitative changes in paxillin distribution but no difference in the actin cytoskeletal structure. Increases in cell size and shape correlate with over-expression of two integrins and the urokinase-type plasminogen activator receptor (uPAR), which is also involved in cell adhesion and is often over-expressed in metastatic cancer cells. These findings demonstrate that gene expression pathways affected by chromatin remodeling enzymes can regulate the physical dimensions of mammalian cell morphology.

Culture Condition-Dependent Senescence-Like Growth Arrest and Immortalization in Rodent Embryo Cells. We investigated the telomerase activity, telomere length, and replicative life span of cells from human embryos and rodent embryos (mouse, rat and Syrian hamster). We used two culture conditions for rodent embryo cells whereby the cells were plated at a density of 2 x 10(5) into a 25-cm(2) flask and subcultured every 3 days or every 10 days. We found that nearly 100% of the cultures of rodent embryo cells become immortal when they are subcultured using the 10-day culture protocol. These rodent embryo cells retain telomerase activity and long telomeres (19-50 kb) in the long-term cultures, whereas human embryo cells rapidly deplete telomerase activity associated with significant shortening of telomeres, and then they senesce. In contrast to the results from 10-day cultures, we found that some mouse cell cultures and most Syrian hamster cell cultures arrest cell growth after 13 and 29 population doublings, respectively, while retaining substantial levels of telomerase activity and experiencing no significant loss of telomeres when the cells were subcultured using the 3-day culture protocol. This growth arrest is phenotypically indistinguishable from cellular senescence. The present results suggest that in rodent cells the onset of senescence-like arrest can be activated without repression of telomerase, and that this activation pathway can be bypassed easily under certain culture conditions, such as the 10-day culture protocol.

We studied the effect of hypoxia on X-ray-induced delayed effects in normal human embryo cells to elucidate the role of oxidative stress in the susceptibility of cells to induction of genetic instability by radiation. We examined X-ray-induced delayed cell death, giant cell formation, and chromosome aberrations under normally oxygenated (20%) and hypoxic (2%) conditions at 28-38 population doublings postirradiation. The results revealed that hypoxia reduced the X-ray-induced delayed effects, suggesting that radiation enhances cellular oxidative stress, which plays a significant role in determining the susceptibility of irradiated cells to genetic instability. The present study emphasizes the biological significance of epigenetic effects, such as oxygen tension, as well as direct DNA damage in the induction of genetic instability by radiation.