Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling

Abstract

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1 Gy of X-rays. While the initial foci size was approximately 0.6 µm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6 µm or more in diameter at 24 h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1 and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1 Gy of X-rays and incubated for 24 h, the grown large phosphorylated ATM foci (≥1.6 µm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6 µm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM 4 by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint.These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.5

Mode of ATM-dependent suppression of chromosome translocation

“…G1 checkpoint is highly sensitive, which can be induced by a single DSB [31]. We previously showed that growth of an IR-induced focus of checkpoint factors, such as Ser1981-phosphorylated ATM and 53BP1, allows damage signal of a single DSB to be amplified to a sufficient level to elicit G1 checkpoint [32]. Therefore, cells harboring translocation can be arrested if they have only one unrepaired DSB.…”

The cellular response to DNA damage: A focus on MDC1 and its interacting proteins

“…As cells recover, the number of foci decreases (signifying repair) and the remaining foci get larger. 4,37 Additionally, several key DDR proteins-NBS1, BRCA1 and 53BP1-are recruited to DSBs even in the absence of histone H2AX. 38 These proteins show early DSB recruitment kinetics in WT or histone H2AX null cells (minutes) but fail to accumulate later on in histone that is suggested to act in resection of complex DSBs.…”

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

“…If this system is active enough, the entry of cells with damaged DNA into the next phase will be arrested until the completion of its repair. However, if the checkpoint system is not activated, the entry transition of cells with critical DNA damage into another phase of the cell cycle can be completed either by fixation of chromosomal disorders or by apoptosis (Costes et al, 2006;Harrison and Haber, 2006;Yamauchi et al, 2008). Therefore, the arrival of a signal to activate the cell cycle checkpoint is a critical event in the development of the complex network of DDR.…”

“…In the scheme involves the activation of cell cycle checkpoint due to DSB. ATM and ATR take signals from sensor proteins capable of recognizing the critical DNA damage, and then phosphorylate the effector kinases Chk1 and Chk2 (checkpoint-kinases 1, 2) (Costes et al, 2006;Yamauchi et al, 2008). Activation of Chk2 occurs through its phosphorylation by ATM kinase at the amino acid T68, followed by oligomerization and autophosphorylation at T383 and T387 (Harrison and Haber, 2006).…”