CtIP links DNA double-strand break sensing to resection.

In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells, we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, a process dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM, and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: it does so by binding to the DNA at DSBs after DSB sensing and ATM activation and then promoting DNA resection, leading to checkpoint activation and HR.

[1]  Wen-Hwa Lee,et al.  CtIP Activates Its Own and Cyclin D1 Promoters via the E2F/RB Pathway during G1/S Progression , 2006, Molecular and Cellular Biology.

[2]  Phang-lang Chen,et al.  Expression of PCNA-binding domain of CtIP, a motif required for CtIP localization at DNA replication foci, causes DNA damage and activation of DNA damage checkpoint , 2009, Cell cycle.

[3]  T. Paull,et al.  Activation and regulation of ATM kinase activity in response to DNA double-strand breaks , 2007, Oncogene.

[4]  J Wade Harper,et al.  The DNA damage response: ten years after. , 2007, Molecular cell.

[5]  Maximina H. Yun,et al.  CtIP-BRCA1 modulates the choice of DNA double-strand break repair pathway throughout the cell cycle , 2009, Nature.

[6]  N. Lakin,et al.  Recruitment of ATR to sites of ionising radiation-induced DNA damage requires ATM and components of the MRN protein complex , 2006, Oncogene.

[7]  R. Greenberg,et al.  Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes. , 2006, Genes & development.

[8]  B. Pulendran,et al.  Ligation-anchored PCR: a simple amplification technique with single-sided specificity. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

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

[10]  Sam A. Johnson,et al.  Rapid activation of ATM on DNA flanking double-strand breaks , 2007, Nature Cell Biology.

[11]  Junjie Chen,et al.  BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. , 2006, Genes & development.

[12]  K. Manova,et al.  Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models , 2005, Nature Cell Biology.

[13]  Stephen P. Jackson,et al.  Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage , 2005, Nature.

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

[15]  C. Smythe,et al.  Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. , 1991, Methods in cell biology.

[16]  N. Curtin,et al.  Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM , 2004, Cancer Research.

[17]  J. M. Boyd,et al.  Interaction between a Cellular Protein That Binds to the C-terminal Region of Adenovirus E1A (CtBP) and a Novel Cellular Protein Is Disrupted by E1A through a Conserved PLDLS Motif* , 1998, The Journal of Biological Chemistry.

[18]  J. Visvader,et al.  Dimerization of CtIP, a BRCA1- and CtBP-interacting Protein, Is Mediated by an N-terminal Coiled-coil Motif* , 2004, Journal of Biological Chemistry.

[19]  S. Smerdon,et al.  A Supramodular FHA/BRCT-Repeat Architecture Mediates Nbs1 Adaptor Function in Response to DNA Damage , 2009, Cell.

[20]  Hiroshi Masuhara,et al.  Three‐dimensional optical trapping and laser ablation of a single polymer latex particle in water , 1991 .

[21]  I. Verma,et al.  Production and purification of lentiviral vectors , 2006, Nature Protocols.

[22]  J. Newport,et al.  Xic1 degradation in Xenopus egg extracts is coupled to initiation of DNA replication. , 2002, Genes & development.

[23]  F. Klein,et al.  A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene , 2007, The EMBO journal.

[24]  Junjie Chen,et al.  N Terminus of CtIP Is Critical for Homologous Recombination-mediated Double-strand Break Repair* , 2009, The Journal of Biological Chemistry.

[25]  S. Lees-Miller,et al.  DNA damage-induced activation of ATM and ATM-dependent signaling pathways. , 2004, DNA repair.

[26]  J. Myers,et al.  Rapid Activation of ATR by Ionizing Radiation Requires ATM and Mre11* , 2006, Journal of Biological Chemistry.

[27]  ATM regulates ATR chromatin loading in response to DNA double-strand breaks , 2006 .

[28]  P. Russell,et al.  Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination. , 2007, Molecular cell.

[29]  Judith L. Campbell,et al.  Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair , 2009, PloS one.

[30]  A. Kumagai,et al.  Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. , 2000, Molecular cell.

[31]  K. Cimprich,et al.  ATR: an essential regulator of genome integrity , 2008, Nature Reviews Molecular Cell Biology.

[32]  Michael W Berns,et al.  Internet‐based robotic laser scissors and tweezers microscopy , 2005, Microscopy research and technique.

[33]  John A. Tainer,et al.  Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair , 2009, Cell.

[34]  B. A. Ballif,et al.  ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage , 2007, Science.

[35]  Jiri Bartek,et al.  ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks , 2006, Nature Cell Biology.

[36]  Xiaohua Wu,et al.  Cell Cycle-dependent Complex Formation of BRCA1·CtIP·MRN Is Important for DNA Double-strand Break Repair* , 2008, Journal of Biological Chemistry.

[37]  L. Zou,et al.  Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks. , 2009, Molecular cell.

[38]  Y. Shiloh,et al.  Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response , 2000, Nature.

[39]  R. Schnabel,et al.  A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination , 2007, The EMBO journal.

[40]  P. Sung,et al.  Mechanism of homologous recombination: mediators and helicases take on regulatory functions , 2006, Nature Reviews Molecular Cell Biology.

[41]  Stephen P. Jackson,et al.  Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair*S⃞ , 2009, Journal of Biological Chemistry.

[42]  P. Russell,et al.  ATM Activation and Its Recruitment to Damaged DNA Require Binding to the C Terminus of Nbs1 , 2005, Molecular and Cellular Biology.

[43]  A. Nussenzweig,et al.  The NBS1-ATM Connection Revisited , 2007, Cell cycle.

[44]  Rodney Rothstein,et al.  At Loose Ends: Resecting a Double-Strand Break , 2009, Cell.

[45]  Wen-Hwa Lee,et al.  CtIP, a Multivalent Adaptor Connecting Transcriptional Regulation, Checkpoint Control and Tumor Suppression , 2006, Cell cycle.

[46]  R. Ghirlando,et al.  Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. , 2007, Molecular cell.

[47]  Eleni P. Mimitou,et al.  Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing , 2008, Nature.

[48]  Junjie Chen,et al.  DNA Damage-Induced Cell Cycle Checkpoint Control Requires CtIP, a Phosphorylation-Dependent Binding Partner of BRCA1 C-Terminal Domains , 2004, Molecular and Cellular Biology.

[49]  Jiri Bartek,et al.  Human CtIP promotes DNA end resection , 2007, Nature.

[50]  K. Ohta,et al.  Molecular Characterization of the Role of the Schizosaccharomyces pombe nip1+/ctp1+ Gene in DNA Double-Strand Break Repair in Association with the Mre11-Rad50-Nbs1 Complex , 2008, Molecular and Cellular Biology.

[51]  Jiri Bartek,et al.  Cell-cycle checkpoints and cancer , 2004, Nature.

[52]  Sang Eun Lee,et al.  Sgs1 Helicase and Two Nucleases Dna2 and Exo1 Resect DNA Double-Strand Break Ends , 2008, Cell.