NFBD 1 / Mdc 1 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 G2-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 ATRdependent 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. (Cancer Res 2005; 65(4): 1158-63)

[1]  N. Lakin,et al.  Recruitment of the Cell Cycle Checkpoint Kinase ATR to Chromatin during S-phase* , 2004, Journal of Biological Chemistry.

[2]  T. Halazonetis,et al.  53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage. , 2003, Cancer research.

[3]  S. Elledge,et al.  Replication protein A-mediated recruitment and activation of Rad17 complexes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Michael G. Sehorn,et al.  Rad51 Recombinase and Recombination Mediators* , 2003, Journal of Biological Chemistry.

[5]  D. Stern,et al.  NFBD1/MDC1 regulates ionizing radiation‐induced focus formation by DNA checkpoint signaling and repair factors , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[6]  Stephen J. Elledge,et al.  Sensing DNA Damage Through ATRIP Recognition of RPA-ssDNA Complexes , 2003, Science.

[7]  Phang-lang Chen,et al.  NFBD1, Like 53BP1, Is an Early and Redundant Transducer Mediating Chk2 Phosphorylation in Response to DNA Damage* , 2003, The Journal of Biological Chemistry.

[8]  Y. Shiloh.  ATM and related protein kinases: safeguarding genome integrity , 2003, Nature Reviews Cancer.

[9]  Junjie Chen,et al.  MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways , 2003, Nature.

[10]  Stephen J. Elledge,et al.  MDC1 is a mediator of the mammalian DNA damage checkpoint , 2003, Nature.

[11]  J. Bartek,et al.  MDC1 is required for the intra-S-phase DNA damage checkpoint , 2003, Nature.

[12]  Phang-lang Chen,et al.  NFBD1, a Novel Nuclear Protein with Signature Motifs of FHA and BRCT, and an Internal 41-Amino Acid Repeat Sequence, Is an Early Participant in DNA Damage Response* , 2003, The Journal of Biological Chemistry.

[13]  M. Kastan,et al.  DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation , 2003, Nature.

[14]  Jiri Bartek,et al.  53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer , 2002, Nature Cell Biology.

[15]  A. Nussenzweig,et al.  DNA damage-induced G2–M checkpoint activation by histone H2AX and 53BP1 , 2002, Nature Cell Biology.

[16]  S. Elledge,et al.  53BP1, a Mediator of the DNA Damage Checkpoint , 2002, Science.

[17]  K. Cimprich,et al.  A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. , 2002, Genes & development.

[18]  D. Toczyski,et al.  A unified view of the DNA-damage checkpoint. , 2002, Current opinion in cell biology.

[19]  Suk-hee Lee,et al.  DNA-dependent protein kinase complex: a multifunctional protein in DNA repair and damage checkpoint. , 2002, Molecules and cells.

[20]  S. Elledge,et al.  Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. , 2002, Genes & development.

[21]  D. Toczyski,et al.  Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. , 2001, Genes & development.

[22]  S. Post,et al.  Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G1/S checkpoint activation in response to DNA damage , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Kunihiro Matsumoto,et al.  Recruitment of Mec1 and Ddc1 Checkpoint Proteins to Double-Strand Breaks Through Distinct Mechanisms , 2001, Science.

[24]  H. Piwnica-Worms,et al.  ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1 , 2001, Molecular and Cellular Biology.

[25]  Xiao-Fan Wang,et al.  ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses , 2001, Nature.

[26]  S. Elledge,et al.  The DNA damage response: putting checkpoints in perspective , 2000, Nature.

[27]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[28]  S. Elledge,et al.  Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. , 2000, Genes & development.

[29]  J. Vialard,et al.  The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1‐dependent hyperphosphorylation and interacts with Rad53 after DNA damage , 1998, The EMBO journal.

[30]  D. Stern,et al.  Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. , 1998, Science.

[31]  E. Rogakou,et al.  DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139* , 1998, The Journal of Biological Chemistry.

[32]  S. Elledge,et al.  The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing , 1994, The Journal of cell biology.

[33]  Phang-lang Chen,et al.  Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation , 1989, Cell.