The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

ABSTRACT Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9−/− and RAD17−/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9−/− and RAD17−/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.

[1]  Alberto Martin,et al.  Immunoglobulin gene conversion: synthesizing antibody diversification and DNA repair. , 2007, DNA repair.

[2]  A. Lu,et al.  The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase , 2007, Nucleic acids research.

[3]  P. V. D. van den Berk,et al.  A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification , 2007, The Journal of experimental medicine.

[4]  M. Neuberger,et al.  Molecular mechanisms of antibody somatic hypermutation. , 2007, Annual review of biochemistry.

[5]  L. Karnitz,et al.  The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. , 2007, Genes & development.

[6]  S. Jentsch,et al.  PCNA, the Maestro of the Replication Fork , 2007, Cell.

[7]  K. Bhakat,et al.  Intracellular trafficking and regulation of mammalian AP-endonuclease 1 (APE1), an essential DNA repair protein. , 2007, DNA repair.

[8]  U. Hübscher,et al.  The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase β in long patch base excision repair , 2007, Nucleic acids research.

[9]  C. Theriot,et al.  The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates NEIL1 glycosylase , 2007, Nucleic acids research.

[10]  A. Yasui,et al.  RAD18 and Poly(ADP-Ribose) Polymerase Independently Suppress the Access of Nonhomologous End Joining to Double-Strand Breaks and Facilitate Homologous Recombination-Mediated Repair , 2007, Molecular and Cellular Biology.

[11]  S. Jentsch,et al.  A Role for PCNA Ubiquitination in Immunoglobulin Hypermutation , 2006, PLoS biology.

[12]  H. Ulrich.  Deubiquitinating PCNA: a downside to DNA damage tolerance , 2006, Nature Cell Biology.

[13]  T. Pandita,et al.  Mammalian Rad9 Plays a Role in Telomere Stability, S- and G2-Phase-Specific Cell Survival, and Homologous Recombinational Repair , 2006, Molecular and Cellular Biology.

[14]  J. Stavnezer.  Faculty Opinions recommendation of Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations. , 2006 .

[15]  K. Nozaki,et al.  Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis. , 2005, Molecular cell.

[16]  T. Shibata,et al.  Rapid generation of specific antibodies by enhanced homologous recombination , 2005, Nature Biotechnology.

[17]  P. Plevani,et al.  The 9-1-1 Checkpoint Clamp Physically Interacts with Polζ and Is Partially Required for Spontaneous Polζ-dependent Mutagenesis in Saccharomyces cerevisiae* , 2005, Journal of Biological Chemistry.

[18]  U. Hübscher,et al.  The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity. , 2005, Journal of molecular biology.

[19]  Y. Taniguchi,et al.  Fen-1 Facilitates Homologous Recombination by Removing Divergent Sequences at DNA Break Ends , 2005, Molecular and Cellular Biology.

[20]  S. Takeda,et al.  Differential and collaborative actions of Rad51 paralog proteins in cellular response to DNA damage , 2005, Nucleic acids research.

[21]  F. Hanaoka,et al.  Different mutation signatures in DNA polymerase eta- and MSH6-deficient mice suggest separate roles in antibody diversification. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[22]  H. Kitao,et al.  Similar Effects of Brca2 Truncation and Rad51 Paralog Deficiency on Immunoglobulin V Gene Diversification in DT40 Cells Support an Early Role for Rad51 Paralogs in Homologous Recombination , 2005, Molecular and Cellular Biology.

[23]  A. Sancar,et al.  The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Neuberger,et al.  Mismatch recognition and uracil excision provide complementary paths to both Ig switching and the A/T-focused phase of somatic mutation. , 2004, Molecular cell.

[25]  Michio Kawasuji,et al.  Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination , 2004, The EMBO journal.

[26]  H. Koyama,et al.  Loss of Nonhomologous End Joining Confers Camptothecin Resistance in DT40 Cells , 2004, Journal of Biological Chemistry.

[27]  B. Beverloo,et al.  Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage‐dependent recombination , 2004, The EMBO journal.

[28]  J. Sale.  Immunoglobulin diversification in DT40: a model for vertebrate DNA damage tolerance. , 2004, DNA repair.

[29]  T. Honjo,et al.  Separate domains of AID are required for somatic hypermutation and class-switch recombination , 2004, Nature Immunology.

[30]  H. Arakawa,et al.  Activation-Induced Cytidine Deaminase Initiates Immunoglobulin Gene Conversion and Hypermutation by a Common Intermediate , 2004, PLoS biology.

[31]  A. Lehmann,et al.  Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. , 2004, Molecular cell.

[32]  O. Matsui,et al.  Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication , 2004, Genes to cells : devoted to molecular & cellular mechanisms.

[33]  U. Hübscher,et al.  Year : 2004 The human Rad 9 / Rad 1 / Hus 1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency : implications for DNA repair , 2006 .

[34]  B. Stillman,et al.  Biochemical Characterization of DNA Damage Checkpoint Complexes: Clamp Loader and Clamp Complexes with Specificity for 5′ Recessed DNA , 2003, PLoS biology.

[35]  M. Yamaizumi,et al.  Multiple roles of Rev3, the catalytic subunit of polζ in maintaining genome stability in vertebrates , 2003, The EMBO journal.

[36]  J. Sale,et al.  Rev1 is essential for DNA damage tolerance and non‐templated immunoglobulin gene mutation in a vertebrate cell line , 2003, The EMBO journal.

[37]  J. Hurwitz,et al.  Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Mihoko Kai,et al.  Checkpoint activation regulates mutagenic translesion synthesis. , 2003, Genes & development.

[39]  Shunichi Takeda,et al.  Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells , 2002, Nature.

[40]  K. Sugimoto,et al.  Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9‐1‐1 and Rad17‐RFC , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[41]  M. Neuberger,et al.  AID Is Essential for Immunoglobulin V Gene Conversion in a Cultured B Cell Line , 2002, Current Biology.

[42]  H. Arakawa,et al.  Requirement of the Activation-Induced Deaminase (AID) Gene for Immunoglobulin Gene Conversion , 2002, Science.

[43]  P. Dhar,et al.  Genetic Analysis of the DNA-dependent Protein Kinase Reveals an Inhibitory Role of Ku in Late S–G2 Phase DNA Double-strand Break Repair* , 2001, The Journal of Biological Chemistry.

[44]  M. Neuberger,et al.  Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation , 2001, Nature.

[45]  D. Schild,et al.  Mutants of the Five Rad51 Paralogs Recombinational Repair in Knockout Chromosome Instability and Defective , 2022 .

[46]  T. Honjo,et al.  Class Switch Recombination and Hypermutation Require Activation-Induced Cytidine Deaminase (AID), a Potential RNA Editing Enzyme , 2000, Cell.

[47]  Yuko Yamaguchi-Iwai,et al.  Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells , 1999, Molecular and Cellular Biology.

[48]  Akira Shinohara,et al.  Rad51‐deficient vertebrate cells accumulate chromosomal breaks prior to cell death , 1998, The EMBO journal.

[49]  Yuko Yamaguchi-Iwai,et al.  Reduced X-Ray Resistance and Homologous Recombination Frequencies in a RAD54−/− Mutant of the Chicken DT40 Cell Line , 1997, Cell.

[50]  B. Bertocci,et al.  Formation of the chicken B-cell repertoire: ontogenesis, regulation of Ig gene rearrangement, and diversification by gene conversion. , 1994, Advances in immunology.

[51]  J. Weill,et al.  A hyperconversion mechanism generates the chicken light chain preimmune repertoire , 1987, Cell.