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PMID: 20729809 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2.

The EMBO journal ·Vol. 29 ·No. 19 ·2010-10-06 ·Pages 3358-69

Mimitou EP, Symington LS

Abstract

In this study, we investigate the interplay between Ku, a central non-homologous end-joining component, and the Mre11-Rad50-Xrs2 (MRX) complex and Sae2, end-processing factors crucial for initiating 5'-3' resection of double-strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Δ sgs1Δ) cannot be suppressed by the yku70Δ mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication-associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end-processing defects established in the sae2Δ sgs1Δ mutant, leading to its lethality.

MeSH Terms
Blotting, Southern DNA Breaks, Double-Stranded DNA Repair DNA-Binding Proteins/metabolism Endodeoxyribonucleases/metabolism Endonucleases/metabolism Exodeoxyribonucleases/metabolism Gamma Rays Multiprotein Complexes/metabolism RecQ Helicases/metabolism Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins/metabolism Telomere/genetics
Chemicals
DNA-Binding Proteins Multiprotein Complexes RAD50 protein, S cerevisiae SAE2 protein, S cerevisiae Saccharomyces cerevisiae Proteins XRS2 protein, S cerevisiae high affinity DNA-binding factor, S cerevisiae Endodeoxyribonucleases Endonucleases Exodeoxyribonucleases MRE11 protein, S cerevisiae exodeoxyribonuclease I SGS1 protein, S cerevisiae RecQ Helicases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Mimitou Eleni P
Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA.
Symington Lorraine S
References (75)
75 references, click to expand
  1. Loss of a yeast telomere: arrest, recovery, and chromosome loss.
    Cell. 1993 Nov 19;75(4):729-39 PMID: 8242745
  2. The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae.
    Mol Cell Biol. 1999 Nov;19(11):7681-7 PMID: 10523656
  3. Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres.
    Mol Cell Biol. 2003 Aug;23(15):5186-97 PMID: 12861005
  4. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination.
    Mol Cell. 2007 Oct 12;28(1):134-46 PMID: 17936710
  5. Multiple pathways regulate 3' overhang generation at S. cerevisiae telomeres.
    Mol Cell. 2009 Jul 10;35(1):70-81 PMID: 19595717
  6. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1.
    Nature. 2004 Oct 21;431(7011):1011-7 PMID: 15496928
  7. EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70Delta mutants.
    Genes Dev. 2002 Aug 1;16(15):1919-33 PMID: 12154123
  8. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs.
    Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10171-6 PMID: 19520826
  9. The DNA-binding protein Hdf1p (a putative Ku homologue) is required for maintaining normal telomere length in Saccharomyces cerevisiae.
    Nucleic Acids Res. 1996 Feb 15;24(4):582-5 PMID: 8604297
  10. The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions.
    Genetics. 2000 Jun;155(2):569-76 PMID: 10835381
  11. Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation.
    Mol Cell Biol. 2005 Jul;25(13):5738-51 PMID: 15964827
  12. RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.
    Nature. 2001 Jul 26;412(6845):456-61 PMID: 11473323
  13. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.
    Mol Cell Biol. 1996 May;16(5):2164-73 PMID: 8628283
  14. Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.
    Genetics. 1998 Oct;150(2):591-600 PMID: 9755192
  15. Nonhomologous end joining in yeast.
    Annu Rev Genet. 2005;39:431-51 PMID: 16285867
  16. The Mre11 nuclease is not required for 5' to 3' resection at multiple HO-induced double-strand breaks.
    Mol Cell Biol. 2004 Nov;24(21):9682-94 PMID: 15485933
  17. The nuclease activity of Mre11 is required for meiosis but not for mating type switching, end joining, or telomere maintenance.
    Mol Cell Biol. 1999 Jan;19(1):556-66 PMID: 9858579
  18. Links between replication and recombination in Saccharomyces cerevisiae: a hypersensitive requirement for homologous recombination in the absence of Rad27 activity.
    Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8263-9 PMID: 11459962
  19. Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia.
    Mol Cell. 2010 Jul 9;39(1):25-35 PMID: 20598602
  20. Telomere maintenance is dependent on activities required for end repair of double-strand breaks.
    Curr Biol. 1998 May 21;8(11):657-60 PMID: 9635193
  21. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.
    Science. 2003 Jun 6;300(5625):1542-8 PMID: 12791985
  22. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase.
    Genes Dev. 2005 Feb 1;19(3):339-50 PMID: 15687257
  23. DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1.
    EMBO J. 2003 Aug 15;22(16):4325-36 PMID: 12912929
  24. The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities.
    Curr Biol. 1998 Jul 2;8(14):831-4 PMID: 9663392
  25. Endonucleolytic processing of covalent protein-linked DNA double-strand breaks.
    Nature. 2005 Aug 18;436(7053):1053-7 PMID: 16107854
  26. Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex.
    Mol Cell. 2007 Nov 30;28(4):638-51 PMID: 18042458
  27. Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining.
    Genetics. 2007 Aug;176(4):2003-14 PMID: 17565964
  28. RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination.
    Cell. 2004 Apr 16;117(2):171-84 PMID: 15084256
  29. Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae.
    DNA Repair (Amst). 2009 Feb 1;8(2):162-9 PMID: 18992851
  30. Systematic genetic analysis with ordered arrays of yeast deletion mutants.
    Science. 2001 Dec 14;294(5550):2364-8 PMID: 11743205
  31. Overlapping functions of the Saccharomyces cerevisiae Mre11, Exo1 and Rad27 nucleases in DNA metabolism.
    Genetics. 2001 Dec;159(4):1423-33 PMID: 11779786
  32. CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle.
    Nature. 2009 May 21;459(7245):460-3 PMID: 19357644
  33. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage.
    Cell. 1998 Aug 7;94(3):399-409 PMID: 9708741
  34. Genetic regulation of telomere-telomere fusions in the yeast Saccharomyces cerevisae.
    Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10854-9 PMID: 12963812
  35. DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray.
    Nat Genet. 2003 Nov;35(3):277-86 PMID: 14566339
  36. Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).
    Genetics. 2002 Jan;160(1):49-62 PMID: 11805044
  37. A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair.
    Cell. 1997 Jan 24;88(2):253-63 PMID: 9008166
  38. The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle.
    EMBO J. 2004 Dec 8;23(24):4868-75 PMID: 15549137
  39. Differential regulation of the cellular response to DNA double-strand breaks in G1.
    Mol Cell. 2008 Apr 11;30(1):73-85 PMID: 18406328
  40. Transformation of intact yeast cells treated with alkali cations.
    J Bacteriol. 1983 Jan;153(1):163-8 PMID: 6336730
  41. Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.
    EMBO J. 2008 Jul 23;27(14):1953-62 PMID: 18596698
  42. The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends.
    J Biol Chem. 2005 Nov 18;280(46):38631-8 PMID: 16162495
  43. Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair.
    Cell. 2008 Oct 3;135(1):97-109 PMID: 18854158
  44. Recruitment and dissociation of nonhomologous end joining proteins at a DNA double-strand break in Saccharomyces cerevisiae.
    Genetics. 2008 Mar;178(3):1237-49 PMID: 18245831
  45. Recombination proteins in yeast.
    Annu Rev Genet. 2004;38:233-71 PMID: 15568977
  46. A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis.
    Mol Cell Biol. 1998 Jan;18(1):260-8 PMID: 9418873
  47. Homologous recombination is required for the viability of rad27 mutants.
    Nucleic Acids Res. 1998 Dec 15;26(24):5589-95 PMID: 9837987
  48. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.
    Genes Dev. 2008 Oct 15;22(20):2767-72 PMID: 18923075
  49. Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.
    J Biol Chem. 2010 Apr 9;285(15):11628-37 PMID: 20150422
  50. Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.
    Genes Dev. 2005 Dec 15;19(24):3055-69 PMID: 16357221
  51. The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together.
    DNA Repair (Amst). 2004 Aug-Sep;3(8-9):845-54 PMID: 15279769
  52. Ku70 corrupts DNA repair in the absence of the Fanconi anemia pathway.
    Science. 2010 Jul 9;329(5988):219-23 PMID: 20538911
  53. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination.
    Genetics. 1992 Nov;132(3):651-64 PMID: 1468624
  54. Break dosage, cell cycle stage and DNA replication influence DNA double strand break response.
    EMBO J. 2008 Jul 9;27(13):1875-85 PMID: 18511906
  55. Enhancement of Saccharomyces cerevisiae end-joining efficiency by cell growth stage but not by impairment of recombination.
    Genetics. 2002 Jul;161(3):1015-27 PMID: 12136007
  56. Mechanism of interaction between Ku protein and DNA.
    J Biol Chem. 1986 Aug 5;261(22):10375-9 PMID: 3015926
  57. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.
    Cell. 2004 Sep 17;118(6):699-713 PMID: 15369670
  58. Ku autoantigen: a multifunctional DNA-binding protein.
    Crit Rev Biochem Mol Biol. 2000;35(1):1-33 PMID: 10755664
  59. Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.
    Genetics. 2001 May;158(1):109-22 PMID: 11333222
  60. Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination.
    Nat Struct Mol Biol. 2007 Jul;14(7):639-46 PMID: 17589524
  61. CDK targets Sae2 to control DNA-end resection and homologous recombination.
    Nature. 2008 Oct 2;455(7213):689-92 PMID: 18716619
  62. Human CtIP mediates cell cycle control of DNA end resection and double strand break repair.
    J Biol Chem. 2009 Apr 3;284(14):9558-65 PMID: 19202191
  63. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.
    Cell. 2008 Sep 19;134(6):981-94 PMID: 18805091
  64. Mechanism and control of meiotic recombination initiation.
    Curr Top Dev Biol. 2001;52:1-53 PMID: 11529427
  65. Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks.
    Mol Cell Biol. 2005 Aug;25(16):7158-69 PMID: 16055725
  66. Interplay of Mre11 nuclease with Dna2 plus Sgs1 in Rad51-dependent recombinational repair.
    PLoS One. 2009;4(1):e4267 PMID: 19165339
  67. Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic recombination.
    Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9356-61 PMID: 19470480
  68. Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae.
    Mol Biol Cell. 2000 Jul;11(7):2221-33 PMID: 10888664
  69. How telomeres are replicated.
    Nat Rev Mol Cell Biol. 2007 Oct;8(10):825-38 PMID: 17885666
  70. DNA end resection: many nucleases make light work.
    DNA Repair (Amst). 2009 Sep 2;8(9):983-95 PMID: 19473888
  71. Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA.
    Mol Cell. 2009 Jan 16;33(1):117-23 PMID: 19150433
  72. Mutations in Mre11 phosphoesterase motif I that impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 complex stability in addition to nuclease activity.
    Genetics. 2005 Dec;171(4):1561-70 PMID: 16143598
  73. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.
    Nature. 2008 Oct 9;455(7214):770-4 PMID: 18806779
  74. The Yku70-Yku80 complex contributes to regulate double-strand break processing and checkpoint activation during the cell cycle.
    EMBO Rep. 2008 Aug;9(8):810-8 PMID: 18600234
  75. The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements.
    Cell. 2002 Jan 25;108(2):183-93 PMID: 11832209
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
1460-2075
DOI
10.1038/emboj.2010.193
Published
2010-10-06
Epub
2010-00-20
Pages
3358-69
Language
English
Region
England
NLM ID
8208664
PMCID
PMC2957202
Subset
IM
Grants
NIGMS NIH HHS · GM041784 · United States
NIGMS NIH HHS · R01 GM041784-21 · United States
NIGMS NIH HHS · R01 GM041784-21S1 · United States
NIGMS NIH HHS · R01 GM041784 · United States
NIGMS NIH HHS · R01 GM041784-22 · United States
External Links
PubMed source DOI: 10.1038/emboj.2010.193 PMC Full Text View journal details
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