Mode of ATM-dependent suppression of chromosome translocation

Abstract

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition orRNAi-mediated knockdown of ATM causes 2~2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

Progression of chromosomal damage induced by etoposide in G2 phase in a DNA-PKcs-deficient context

“…This result agrees with a previous study of Yamauchi et al (2011), which indicates that DNA-PKcs has some role in suppressing the occurrence of dicentric chromosomes following gamma irradiation in normal human fibroblasts.…”

“…d Representative images of HeLa BN cells with γH2AX foci (FITC) in the main nuclei following ETO treatment for 1 h. DNA was stained with DAPI. Scale bar, 10 μm translocations and dicentric chromosomes; it has been established that in G2-irradiated cells, translocations and dicentrics are generated by identical mechanism (Yamauchi et al 2011). First, we assessed the phosphorylation of H3 at serine 10 to identify mitotic cells.…”

DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

“…Medium was refreshed 12 h post IR, and cells were incubated with 4 μM APH, 0.1 mg/mL Colcemid and 600 μM UCN-01, which abolishes G2/M checkpoint arrest, 12–16 h post-IR to collect irradiated G2 cells in mitosis. Metaphase spread and giemsa staining were as described (Yamauchi et al, 2011). …”

Effect of X-Irradiation at Different Stages in the Cell Cycle on Individual Cell–Based Kinetics in an Asynchronous Cell Population

“…Therefore, we speculated that cells carrying a wild-type p53 gene with Fucci probes may exhibit elongation of red phase, representing G1 arrest. Because appropriate regulation of p53 expression was very difficult in this system, we performed further analysis using BJ1-hTERT-Fucci cells, which were established from h-TERT–immortalized normal human diploid foreskin fibroblasts (BJ-hTERT) with wild-type p53 function [27]. In an unirradiated condition, green cells entered M phase first, whereas red cells entered M phase after almost all the green cells had done so (Fig 8A), like Fucci-HeLa cells (Fig 1D).…”

“…In the Fucci system, cells emitting red, green, or orange (red plus green) fluorescence correspond to G1, S/G2/M, and early S phase, respectively [24]. BJ1-hTERT-Fucci cells expressing the Fucci probes were established from h-TERT-immortalized normal human diploid foreskin fibroblasts (BJ-hTERT) [27] using a previously described method [19]. All cell lines were maintained at 37°C in a 5% CO 2 humidified atmosphere in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum.…”