A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs؊/؊ and Atm؊/؊ cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs؊/؊ cells and severely compromised in Atm؊/؊ cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm؊/؊ fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm؊/؊ fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.DNA double-strand breaks (DSBs) 1 are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination (1). A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage (2). H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells (3) resulting in discrete ␥-H2AX (phosphorylated-H2AX) foci at the DNA damage sites (4). In experiments involving the use of "laser scissors" to introduce breaks into living cells, ␥-H2AX foci localized specifically with the laser path through the cell nuclei clearly demonstrating that H2AX phosphorylation is specific to the sites of DNA damage (4, 5). H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells (6) and meiotic recombination in mice (7). Phosphorylation of yeast H2A at serine 129 (homologous to serine 139 of mammalian H2AX) causes chromatin decondensation and is required for efficient DNA double-strand break repair (8). In mammals, phosphorylation of H2AX appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage (5, 9).As H2AX phosphorylation plays a very early and important role in the cellular response to DNA double-strand breaks, it is important to specifically identify the kinase(s) involved in this event. Members of the PI 3-kinase family, including DNA-PK (DNA-dependent protein kinase...

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.

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Summary The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary post-natal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen rich postnatal environment is the upstream signal that results in cell cycle arrest of cardiomyocytes. Here we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, while hyperoxemia and ROS generators shorten it. These findings uncover a previously unrecognized protective mechanism that mediates cardiomyocyte cell cycle arrest in exchange for utilization of oxygen dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be important component of cardiomyocyte proliferation-based therapeutic approaches.

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.