Mdc1 couples DNA double‐strand break recognition by Nbs1 with its H2AX‐dependent chromatin retention

Mdc1/NFBD1 controls cellular responses to DNA damage, in part via interacting with the Mre11–Rad50–Nbs1 complex that is involved in the recognition, signalling, and repair of DNA double‐strand breaks (DSBs). Here, we show that in live human cells, the transient interaction of Nbs1 with DSBs and its phosphorylation by ATM are Mdc1‐independent. However, ablation of Mdc1 by siRNA or mutation of the Nbs1's FHA domain required for Mdc1 binding reduced the affinity of Nbs1 for DSB‐flanking chromatin and caused aberrant pan‐nuclear dispersal of Nbs1. This occurred despite normal phosphorylation of H2AX, indicating that lack of Mdc1 does not impair this DSB‐induced chromatin change, but rather precludes the sustained engagement of Nbs1 with these regions. Mdc1 (but not Nbs1) became partially immobilized to chromatin after DSB generation, and siRNA‐mediated depletion of H2AX prevented such relocalization of Mdc1 and uncoupled Nbs1 from DSB‐flanking chromatin. Our data suggest that Mdc1 functions as an H2AX‐dependent interaction platform enabling a switch from transient, Mdc1‐independent recruitment of Nbs1 to DSBs towards sustained, Mdc1‐dependent interactions with the surrounding chromosomal microenvironment.

[1]  K. Tanimoto,et al.  NBS1 Localizes to γ-H2AX Foci through Interaction with the FHA/BRCT Domain , 2002, Current Biology.

[2]  R. T. Bree,et al.  The MRN complex: coordinating and mediating the response to broken chromosomes , 2003, EMBO reports.

[3]  J. Petrini,et al.  The cellular response to DNA double-strand breaks: defining the sensors and mediators. , 2003, Trends in cell biology.

[4]  R. Bonner,et al.  Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks , 2003, Nature Cell Biology.

[5]  M. Gatei,et al.  ATM-dependent phosphorylation of nibrin in response to radiation exposure , 2000, Nature Genetics.

[6]  K. Tanimoto,et al.  NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. , 2002, Current biology : CB.

[7]  Bo Xu,et al.  ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway , 2000, Nature.

[8]  Xingzhi Xu,et al.  NFBD1/KIAA0170 Is a Chromatin-associated Protein Involved in DNA Damage Signaling Pathways* , 2003, The Journal of Biological Chemistry.

[9]  J. Bartek,et al.  Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation , 2004, Oncogene.

[10]  K. Cerosaletti,et al.  Nibrin Forkhead-associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation* , 2003, Journal of Biological Chemistry.

[11]  J. Bartek,et al.  Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage , 2003, Nature Cell Biology.

[12]  Phang-lang Chen,et al.  The Nijmegen Breakage Syndrome Protein Is Essential for Mre11 Phosphorylation upon DNA Damage* , 1999, The Journal of Biological Chemistry.

[13]  Yair Andegeko,et al.  Requirement of the MRN complex for ATM activation by DNA damage , 2003, The EMBO journal.

[14]  J. Petrini,et al.  DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex , 2001, Molecular and Cellular Biology.

[15]  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.

[16]  C. Lilley,et al.  The Mre11 complex is required for ATM activation and the G2/M checkpoint , 2003, The EMBO journal.

[17]  J. Bartek,et al.  The DNA damage-dependent intra–S phase checkpoint is regulated by parallel pathways , 2002, Nature Genetics.

[18]  Bo Xu,et al.  Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage. , 2003, Molecular cancer research : MCR.

[19]  S. T. Kim,et al.  Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members* , 1999, The Journal of Biological Chemistry.

[20]  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.

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

[22]  Bruce Stillman,et al.  Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis , 2000, Molecular and Cellular Biology.

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

[24]  Junjie Chen,et al.  Mediator of DNA Damage Checkpoint Protein 1 Regulates BRCA1 Localization and Phosphorylation in DNA Damage Checkpoint Control* , 2003, The Journal of Biological Chemistry.

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

[26]  Wim Vermeulen,et al.  Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage , 2002, The EMBO journal.

[27]  S. Jackson,et al.  The MRE11 complex: at the crossroads of DNA repair and checkpoint signalling , 2002, Nature Reviews Molecular Cell Biology.

[28]  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.

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

[30]  C. Limoli,et al.  A new method for introducing double-strand breaks into cellular DNA. , 1993, Radiation research.

[31]  Shinya Matsuura,et al.  Nijmegen breakage syndrome gene, NBS1, and molecular links to factors for genome stability , 2002, Oncogene.

[32]  K. Cerosaletti,et al.  Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization , 2001, Molecular and Cellular Biology.

[33]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[34]  Y. Shiloh,et al.  Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products , 2000, Nature.

[35]  N. Nomura,et al.  NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain. , 2000, DNA and cell biology.

[36]  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.

[37]  R. Painter,et al.  Radiosensitivity in ataxia-telangiectasia: a new explanation. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[38]  E. Rogakou,et al.  Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo , 1999, The Journal of cell biology.