Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of Chk2 (at thr-68) and p53 (at ser-15). p53 phosphorylation was reduced by the ATM inhibitor caffeine, and H2AX phosphorylation was greatly reduced in AT cells, confirming that phosphorylation was primarily ATM-dependent. We also examined the effects of BrdU photolysis on the major cellular signaling ERK pathways. Interestingly, low-dose (≤ 2 Gy-equivalents) BrdU photolysis stimulated ERK1/2 phosphorylation whereas higher doses (≥ 5 Gy eq.) resulted in Em1/2 dephosphorylation. ERK1/2 phosphorylation was ATM-dependent, whereas dephosphorylation was ATM-independent and DSBs dose-dependent. Thus ERK1/2 appear to be both positively and negatively regulated by ATM depending on the severity of the insult to DNA. In summary, few DSBs trigger ATM-dependent ERK1/2 pro-survival signals whereas more DSBs result in ERK1/2 dephosphorylation consistent with a switch from pro-survival to anti-survival signaling that might affect DSBs repair.