NFBD1/MDC1 regulates ionizing radiation-induced focus formation by DNA checkpoint signaling and repair factors.
NFBD1/MDC1 (mediator of DNA damage checkpoint 1) is a nuclear factor with an amino-terminal FHA (forkhead-associated) domain and a tandem repeat of BRCT (breast cancer susceptibility gene-1 carboxyl terminus) domains. We have previously shown that NFBD1 is an early participant in DNA damage signaling pathways and that ionizing radiation-induced nuclear foci (IRIF) of NFBD1 colocalize with several DNA checkpoint signaling and repair factors. We report here that NFBD1 physically associates with ATM, p53, components of the MRE11-RAD50-NBS1 (MRN) complex, and gamma-H2AX. An overexpressed FHA domain-containing fragment of NFBD1 binds to endogenous NFBD1 and components of the MRN complex, but not to gamma-H2AX. This fragment interferes with IRIF formation by endogenous NFBD1, MRE11, or NBS1. A BRCT domain-containing fragment of NFBD1 binds to gamma-H2AX and 53BP1, but not to components of the MRN complex, and abolishes IRIF formation by NFBD1, MRE11, NBS1, 53BP1, CHK2 phospho-T68, gamma-H2AX, and possible ATM/ATR substrates recognized by anti-phospho-SQ/TQ antibody. These results suggest that NFBD1 is an ATM/ATR-dependent organizer that recruits DNA checkpoint signaling and repair proteins to the sites of DNA damage.
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011839:Radiation, Ionizing; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D015534:Trans-Activators