Breaks Invisible to the DNA Damage Response Machinery Accumulate in ATM-Deficient Cells

After irradiation, ATM defective cells accumulate unrepaired double strand breaks (DSBs) for several cell divisions. At the chromosome level, unresolved DSBs appear as chromosome breaks that can be efficiently scored by using telomeric and mFISH probes. H2AX is immediately activated by ATM in response to DNA damage and its phosphorylated form, gamma H2AX, flanks the DSB through several megabases. The gamma H2AX-labeling status of broken chromosome ends was analyzed in AT cells to check whether the DNA damage response was accurately taking place in these persistent DSBs. The results show that one quarter of the scored breaks are devoid of gamma H2AX foci in metaphase spreads from ATM-deficient cells, and this fraction is significantly higher than in normal cells (chi(2) < 0.05). Accumulation of sensor and repair proteins at damaged sites is a key event in the cellular response to DSBs, so MREII labeling at broken ends was also analyzed. While all gamma H2AX foci scored at visible broken ends colocalize with MRE11 foci, all gamma H2AX-unlabeled breaks are also devoid of MRE11-labeling. The present results suggest that a significant subset of the AT long-lived DSBs may persist as invisible DSBs due to deficient detection by the DNA damage repair machinery. Eventually the properly signaled DSBs will be repaired while invisible breaks may indefinitely accumulate; most probably contributing to the AT cells' well known genomic instability. (C) 2009 Wiley-Liss, Inc.