DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

[1]  H. Pickett,et al.  NuRD–ZNF827 recruitment to telomeres creates a molecular scaffold for homologous recombination , 2014, Nature Structural &Molecular Biology.

[2]  K. Savage,et al.  BRCA1, a ‘complex’ protein involved in the maintenance of genomic stability , 2015, The FEBS journal.

[3]  M. Nussenzweig,et al.  53BP1 facilitates long-range DNA end-joining during V(D)J recombination , 2008, Nature.

[4]  Kurt Hornik,et al.  Implementing a Class of Permutation Tests: The coin Package , 2008 .

[5]  A. D’Andrea,et al.  Repair Pathway Choices and Consequences at the Double-Strand Break. , 2016, Trends in cell biology.

[6]  Jürgen Cox,et al.  Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links , 2015, Science.

[7]  Peter Bouwman,et al.  REV7 counteracts DNA double-strand break resection and affects PARP inhibition , 2015, Nature.

[8]  D. Adams,et al.  53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers , 2010, Nature Structural &Molecular Biology.

[9]  José A. Dianes,et al.  2016 update of the PRIDE database and its related tools , 2016, Nucleic Acids Res..

[10]  T. Lange,et al.  Telomeric 3′ Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST , 2012, Cell.

[11]  Pedro M. Valero-Mora,et al.  ggplot2: Elegant Graphics for Data Analysis , 2010 .

[12]  T. Halazonetis,et al.  P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks , 2000, The Journal of cell biology.

[13]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[14]  Wolfgang Viechtbauer,et al.  Conducting Meta-Analyses in R with the metafor Package , 2010 .

[15]  H. Ji,et al.  The mTOR-S6K Pathway Links Growth Signaling to DNA Damage Response by Targeting RNF168 , 2017, Nature Cell Biology.

[16]  Ellen T. Gelfand,et al.  The Genotype-Tissue Expression (GTEx) project , 2013, Nature Genetics.

[17]  R. Verdun,et al.  Defective Telomere Lagging Strand Synthesis in Cells Lacking WRN Helicase Activity , 2004, Science.

[18]  Junjie Chen.  Faculty Opinions recommendation of DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. , 2015 .

[19]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[20]  S. West,et al.  The SMX DNA Repair Tri-nuclease , 2017, Molecular cell.

[21]  S. Carr,et al.  Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging , 2013, Science.

[22]  Sudhir Kumar,et al.  TimeTree: A Resource for Timelines, Timetrees, and Divergence Times. , 2017, Molecular biology and evolution.

[23]  Facundo D. Batista,et al.  RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection , 2013, Molecular cell.

[24]  F. Alt,et al.  Evolution of the immunoglobulin heavy chain class switch recombination mechanism. , 2007, Advances in immunology.

[25]  Alan Ashworth,et al.  PARP inhibitors: Synthetic lethality in the clinic , 2017, Science.

[26]  T. de Lange,et al.  Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2. , 2008, Genes & development.

[27]  Wilhelm Palm,et al.  How shelterin protects mammalian telomeres. , 2008, Annual review of genetics.

[28]  M. Mann,et al.  HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment , 2013, The EMBO journal.

[29]  Thomas Helleday,et al.  Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase , 2005, Nature.

[30]  D. Durocher,et al.  MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5′ end-resection , 2015, Nature.

[31]  Ulrich Bodenhofer,et al.  msa: an R package for multiple sequence alignment , 2015, Bioinform..

[32]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[33]  P. Calsou,et al.  Loss of BRCA1 impairs centromeric cohesion and triggers chromosomal instability , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[34]  Anushya Muruganujan,et al.  PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements , 2016, Nucleic Acids Res..

[35]  Alan Ashworth,et al.  Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy , 2005, Nature.

[36]  Erik L. L. Sonnhammer,et al.  InParanoid 8: orthology analysis between 273 proteomes, mostly eukaryotic , 2014, Nucleic Acids Res..

[37]  J. Lawrence,et al.  BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin , 2006, The Journal of cell biology.

[38]  Sandy Chang,et al.  TERRA and hnRNPA1 Orchestrate an RPA-to-POT1 Switch on Telomeric Single-Stranded DNA , 2010, Nature.

[39]  J. Lingner,et al.  The human CST complex is a terminator of telomerase activity , 2012, Nature.

[40]  Hashimoto,et al.  Purification, crystallization and initial X-ray diffraction study of human REV7 in complex with a REV3 fragment , 2009 .

[41]  M. Nussenzweig,et al.  53BP1 is required for class switch recombination , 2004, The Journal of cell biology.

[42]  Jeremy M. Stark,et al.  53BP1 Inhibits Homologous Recombination in Brca1-Deficient Cells by Blocking Resection of DNA Breaks , 2010, Cell.

[43]  T. Honjo,et al.  High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. , 1996, International immunology.

[44]  A. Aguilera,et al.  Transcription as a Threat to Genome Integrity. , 2016, Annual review of biochemistry.

[45]  J. Bartek,et al.  The DNA-damage response in human biology and disease , 2009, Nature.

[46]  Junjie Chen,et al.  Tumor Suppressor P53 Binding Protein 1 (53bp1) Is Involved in DNA Damage–Signaling Pathways , 2001, The Journal of cell biology.

[47]  S. Bekker-Jensen,et al.  Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1 , 2005, The Journal of cell biology.

[48]  Daniel Durocher,et al.  The control of DNA repair by the cell cycle , 2016, Nature Cell Biology.

[49]  Cathy H. Wu,et al.  UniProt: the Universal Protein knowledgebase , 2004, Nucleic Acids Res..

[50]  F. Alt,et al.  53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination , 2004, Nature Immunology.

[51]  Hadley Wickham,et al.  ggplot2 - Elegant Graphics for Data Analysis (2nd Edition) , 2017 .

[52]  E. Lazzerini Denchi,et al.  TZAP: A telomere-associated protein involved in telomere length control , 2017, Science.

[53]  Kara Dolinski,et al.  The BioGRID interaction database: 2017 update , 2016, Nucleic Acids Res..

[54]  K. Riabowol,et al.  REAP: A two minute cell fractionation method , 2010, BMC Research Notes.

[55]  N. Mailand,et al.  ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA , 2013, Cell.

[56]  J. Lukas,et al.  53BP1 fosters fidelity of homology-directed DNA repair , 2016, Nature Structural &Molecular Biology.

[57]  Adam P. Rosebrock,et al.  A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. , 2013, Molecular cell.

[58]  Peer Bork,et al.  Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees , 2016, Nucleic Acids Res..

[59]  A. Zarrin,et al.  Origin of Immunoglobulin Isotype Switching , 2012, Current Biology.