Relative contribution of four nucleases, CtIP, Dna2, Exo1 and Mre11, to the initial step of DNA double‐strand break repair by homologous recombination in both the chicken DT40 and human TK6 cell lines

Homologous recombination (HR) is initiated by double‐strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3′ single‐strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long‐range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1‐deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.

[1]  DNA double strand break repair pathway shoice is directed by distinct MRE 11 nuclease activities , 2017 .

[2]  Mohiuddin,et al.  Smarcal1 promotes double-strand-break repair by nonhomologous end-joining , 2015, Nucleic acids research.

[3]  Nguyen Ngoc Hoa,et al.  BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination , 2015, PloS one.

[4]  P. Cejka,et al.  Sae2 promotes dsDNA endonuclease activity within Mre11–Rad50–Xrs2 to resect DNA breaks , 2014, Nature.

[5]  M. Steinlage,et al.  Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1 , 2014, The Journal of cell biology.

[6]  Hailong Wang,et al.  Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. , 2014, Molecular cell.

[7]  Jing He,et al.  CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity. , 2014, Molecular cell.

[8]  M. Honma,et al.  Tracing the fates of site-specifically introduced DNA adducts in the human genome. , 2014, DNA repair.

[9]  John A Tainer,et al.  DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. , 2014, Molecular cell.

[10]  Yi Zhou,et al.  Quantitation of DNA double-strand break resection intermediates in human cells , 2013, Nucleic acids research.

[11]  M. Steinlage,et al.  Polo like kinase 3 regulates CtIP during DNA double strand break repair in G 1 , 2014 .

[12]  T. Kunkel,et al.  Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes , 2013, Proceedings of the National Academy of Sciences.

[13]  Masato T. Kanemaki,et al.  The Elg1 replication factor C-like complex functions in PCNA unloading during DNA replication. , 2013, Molecular cell.

[14]  S. Cockell,et al.  MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA , 2012, Biology Open.

[15]  Ju-Seog Lee,et al.  Human nuclease/helicase DNA2 alleviates replication stress by promoting DNA end resection. , 2012, Cancer research.

[16]  L. Symington,et al.  Double-strand break end resection and repair pathway choice. , 2011, Annual review of genetics.

[17]  W. Sakai,et al.  The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination , 2011, PLoS genetics.

[18]  B. Wold,et al.  Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint , 2011, Cell cycle.

[19]  A. Egashira,et al.  Double-Strand Break Repair-Independent Role for BRCA2 in Blocking Stalled Replication Fork Degradation by MRE11 , 2011, Cell.

[20]  M. Seki,et al.  The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells. , 2011, Biochimica et biophysica acta.

[21]  J. Petrini,et al.  The MRE11 complex: starting from the ends , 2011, Nature Reviews Molecular Cell Biology.

[22]  K. Bloom,et al.  Chromosome integrity at a double-strand break requires exonuclease 1 and MRX. , 2011, DNA repair.

[23]  M. Seki,et al.  The N-terminal region of RECQL 4 lacking the helicase domain is both essential and suf fi cient for the viability of vertebrate cells Role of the N-terminal region of RECQL 4 in cells , 2011 .

[24]  T. Paull,et al.  Mre11–Rad50–Xrs2 and Sae2 promote 5’ strand resection of DNA double-strand breaks , 2010, Nature Structural &Molecular Biology.

[25]  Eleni P. Mimitou,et al.  Ku prevents Exo1 and Sgs1‐dependent resection of DNA ends in the absence of a functional MRX complex or Sae2 , 2010, The EMBO journal.

[26]  Yu Zhang,et al.  Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks , 2010, The EMBO journal.

[27]  Rohit Prakash,et al.  Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae , 2010, Nature.

[28]  S. Kowalczykowski,et al.  DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2 , 2010, Nature.

[29]  Y. Pommier,et al.  Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair , 2010, PLoS genetics.

[30]  T. Paull,et al.  Saccharomyces cerevisiae Mre 11 / Rad 50 / Xrs 2 and Ku proteins regulate association of Exo 1 and Dna 2 with DNA breaks , 2010 .

[31]  Eleni P. Mimitou.  Ku prevents Exo 1 and Sgs 1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae 2 , 2010 .

[32]  J. Kobayashi,et al.  Bisbenzamidine derivative, pentamidine represses DNA damage response through inhibition of histone H2A acetylation , 2010, Molecular Cancer.

[33]  Tatsuo Fukagawa,et al.  An auxin-based degron system for the rapid depletion of proteins in nonplant cells , 2009, Nature Methods.

[34]  P. Pasero,et al.  The MRX complex stabilizes the replisome independently of the S phase checkpoint during replication stress , 2009, The EMBO journal.

[35]  David J. Chen,et al.  Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1. , 2009, Biochemical and biophysical research communications.

[36]  M. J. Neale,et al.  Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. , 2009, Molecular cell.

[37]  J. Sale,et al.  Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes , 2009, PLoS genetics.

[38]  C. Wyman,et al.  RAD50, an SMC family member with multiple roles in DNA break repair: how does ATP affect function? , 2009, Chromosome Research.

[39]  S. Jackson,et al.  DNA helicases Sgs1 and BLM promote DNA double-strand break resection. , 2008, Genes & development.

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

[41]  D. Ferguson,et al.  Mre11 Nuclease Activity Has Essential Roles in DNA Repair and Genomic Stability Distinct from ATM Activation , 2008, Cell.

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

[43]  M. Seki,et al.  RecQ family helicases in genome stability: Lessons from gene disruption studies in DT40 cells , 2008, Cell cycle.

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

[45]  Raffaella Corvi,et al.  How to reduce false positive results when undertaking in vitro genotoxicity testing and thus avoid unnecessary follow-up animal tests: Report of an ECVAM Workshop. , 2007, Mutation research.

[46]  Judith L. Campbell,et al.  Biochemical analysis of human Dna2 , 2006, Nucleic acids research.

[47]  V. Schreiber,et al.  Parp‐1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells , 2006, The EMBO journal.

[48]  L. Symington,et al.  Mutations in Mre11 Phosphoesterase Motif I That Impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 Complex Stability in Addition to Nuclease Activity , 2005, Genetics.

[49]  M. J. Neale,et al.  Endonucleolytic processing of covalent protein-linked DNA double-strand breaks , 2005, Nature.

[50]  L. Symington,et al.  The Mre11 Nuclease Is Not Required for 5′ to 3′ Resection at Multiple HO-Induced Double-Strand Breaks , 2004, Molecular and Cellular Biology.

[51]  S. Takeda,et al.  Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. , 2004, DNA repair.

[52]  P. Sung,et al.  Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells. , 2004, Genetics.

[53]  M. Ikeguchi,et al.  Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast , 2003, Proceedings of the National Academy of Sciences of the United States of America.

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

[55]  A. Matsuura,et al.  Competition between the Rad50 Complex and the Ku Heterodimer Reveals a Role for Exo1 in Processing Double-Strand Breaks but Not Telomeres , 2003, Molecular and Cellular Biology.

[56]  Wensheng Wang,et al.  Functional Relation among RecQ Family Helicases RecQL1, RecQL5, and BLM in Cell Growth and Sister Chromatid Exchange Formation , 2003, Molecular and Cellular Biology.

[57]  M. Honma,et al.  Deletion, rearrangement, and gene conversion; genetic consequences of chromosomal double‐strand breaks in human cells , 2003, Environmental and molecular mutagenesis.

[58]  Y. Furuichi,et al.  Werner and Bloom helicases are involved in DNA repair in a complementary fashion , 2002, Oncogene.

[59]  D. Gordenin,et al.  The Mre11 Complex Is Required for Repair of Hairpin-Capped Double-Strand Breaks and Prevention of Chromosome Rearrangements , 2002, Cell.

[60]  C. Dekker,et al.  Human Rad50/Mre11 is a flexible complex that can tether DNA ends. , 2001, Molecular cell.

[61]  P. Sung,et al.  DNA Structure-specific Nuclease Activities in theSaccharomyces cerevisiae Rad50·Mre11 Complex* , 2001, The Journal of Biological Chemistry.

[62]  S. Loeillet,et al.  Links between replication and recombination in Saccharomyces cerevisiae: A hypersensitive requirement for homologous recombination in the absence of Rad27 activity , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[63]  伊東 淳.  Expression of full-length NBS1 protein restores normal radiation responses in cells from Nijmegen breakage syndrome patients , 2000 .

[64]  T. Stankovic,et al.  The DNA Double-Strand Break Repair Gene hMRE11 Is Mutated in Individuals with an Ataxia-Telangiectasia-like Disorder , 1999, Cell.

[65]  T Yagi,et al.  Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells , 1999, The EMBO journal.

[66]  J. R. Ferguson,et al.  The Nuclease Activity of Mre11 Is Required for Meiosis but Not for Mating Type Switching, End Joining, or Telomere Maintenance , 1999, Molecular and Cellular Biology.

[67]  B. Nelms,et al.  Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11. , 1998, Genetics.

[68]  Y. Yamaguchi-Iwai,et al.  Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells , 1998, The EMBO journal.

[69]  T. Paull,et al.  The 3' to 5' exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. , 1998, Molecular cell.

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

[71]  T. Sofuni,et al.  A comparative study of TK6 human lymphoblastoid and L5178Y mouse lymphoma cell lines in the in vitro micronucleus test. , 1996, Mutation research.

[72]  Jean-Marie Buerstedde,et al.  Increased ratio of targeted to random integration after transfection of chicken B cell lines , 1991, Cell.