NFBD1/Mdc1 mediates ATR-dependent DNA damage response.
Budding yeast Rad9 (scRad9) plays a central role in mediating Mec1-dependent phosphorylation by recruiting its downstream substrates. The human scRad9 orthologues 53BP1 and NFBD1 associate with ionizing radiation-induced foci (IRIF) at sites of DNA repair. RNAi-based gene silencing of 53BP1 or NFBD1 has shown impaired phosphorylation of SQ/TQ [ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) substrates] at IRIF, intra-S, and G(2)-M checkpoints and has thereby revealed essential roles for 53BP1 and NFBD1 in the DNA damage signaling pathway. Whether 53BP1 and NFBD1 are required for activation of kinases and/or for recruitment of substrates at IRIF, however, is not clear. Here we show that both 53BP1 and NFBD1 are required for recruitment of ATR to DNA damage sites, as well as for ATR-dependent phosphorylation in response to DNA damage. NFBD1 is not required for ssDNA generation at DNA damage sites and is not recruited by replication protein A (RPA)-coated ssDNA. We therefore show that recruitment of NFBD1 and/or 53BP1, the factors downstream of H2AX, is independent of ssDNA generation and RPA coating, whereas both ssDNA and RPA coating play key roles in regulation of the ATR-dependent pathway. These novel findings help clarify where NFBD1 functions in DNA damage early responses.
Peng
Aimin
A
Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Chen
Phang-Lang
PL
D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D051716:Replication Protein A; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology*; D000071857:Tumor Suppressor p53-Binding Protein 1
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; C497167:RPA1 protein, human; C113575:Rad17 protein, human; D051716:Replication Protein A; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases