Signaling pathways leading to ATM activation

53Bp1, NFBD1/MDC1, and Nbs1 are rapidly recruited to sites of DNA double strand breaks (DSBs) where we hypothesize they function to activate the ATM kinase. To test this hypothesis, we suppressed 53Bp1 and NFBD1/MDC1 expression by siRNA and monitored ATM autophosphorylation at Ser1981 and phosphorylation of ATM substrates as markers for ATM activation. We demonstrated that 53Bp1 functions as a DNA damage checkpoint protein and is required for cell cycle arrest at the G2/M checkpoint following irradiation. Additionally, 53Bp1 was required for a subset of ATM-dependent phosphorylation events at the sites of DSBs. Suppression of NFBD1/MDC1 led to decreased ATM activation and decreased phosphorylation of ATM substrates. This phenotype was identical to that observed in cells with defective Nbs1 function and is consistent with recent observations identifying NFBD1/MDC1 as a component of the Mre11-Rad50-Nbs1 protein complex. In cells with wild-type Nbs1, suppression of 53Bp1 expression had no effect on ATM activation, but was associated with increased recruitment of NFBD1/MDC1 and Nbs1 to sites of DNA breaks, suggesting that decreased 53Bp1 function might be compensated for by increased NFBD1/MDC1 and Nbs1 activity. Accordingly, in cells with mutant Nbs1, suppression of 53Bp1 led to decreased ATM activation and decreased phosphorylation of ATM substrates. We conclude that DNA DSBs activate ATM through at least two independent pathways involving 53Bp1 and NFBD1/MDC1-Nbs1, respectively