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Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair


Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.


Vyšlo v časopise: Collaborative Action of Brca1 and CtIP in Elimination of Covalent Modifications from Double-Strand Breaks to Facilitate Subsequent Break Repair. PLoS Genet 6(1): e32767. doi:10.1371/journal.pgen.1000828
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1000828

Souhrn

Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.


Zdroje

1. BarberLJ

BoultonSJ

2006 BRCA1 ubiquitylation of CtIP: Just the tIP of the iceberg? DNA Repair (Amst) 5 1499 1504

2. NealeMJ

PanJ

KeeneyS

2005 Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436 1053 1057

3. SartoriAA

LukasC

CoatesJ

MistrikM

FuS

2007 Human CtIP promotes DNA end resection. Nature 450 509 514

4. PrinzS

AmonA

KleinF

1997 Isolation of COM1, a new gene required to complete meiotic double-strand break-induced recombination in Saccharomyces cerevisiae. Genetics 146 781 795

5. McKeeAH

KlecknerN

1997 A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2. Genetics 146 797 816

6. LengsfeldBM

RattrayAJ

BhaskaraV

GhirlandoR

PaullTT

2007 Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex. Mol Cell 28 638 651

7. HuertasP

JacksonSP

2009 Human CtIP mediates cell cycle control of DNA end resection and double strand break repair. J Biol Chem 284 9558 9565

8. HuertasP

Cortes-LedesmaF

SartoriAA

AguileraA

JacksonSP

2008 CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature 455 689 692

9. MikiY

SwensenJ

Shattuck-EidensD

FutrealPA

HarshmanK

1994 A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266 66 71

10. WangB

MatsuokaS

BallifBA

ZhangD

SmogorzewskaA

2007 Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316 1194 1198

11. ChenL

NieveraCJ

LeeAY

WuX

2008 Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair. J Biol Chem 283 7713 7720

12. YuX

FuS

LaiM

BaerR

ChenJ

2006 BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. Genes Dev 20 1721 1726

13. BermejoR

DoksaniY

CapraT

KatouYM

TanakaH

2007 Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21 1921 1936

14. WangJC

2002 Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3 430 440

15. PommierY

2006 Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6 789 802

16. AndohT

IshidaR

1998 Catalytic inhibitors of DNA topoisomerase II. Biochim Biophys Acta 1400 155 171

17. NitissJL

2009 Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9 338 350

18. AdachiN

SuzukiH

IiizumiS

KoyamaH

2003 Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage. J Biol Chem 278 35897 35902

19. AdachiN

SoS

KoyamaH

2004 Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase I-mediated DNA damage. J Biol Chem 279 37343 37348

20. HartsuikerE

NealeMJ

CarrAM

2009 Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA. Mol Cell 33 117 123

21. YamazoeM

SonodaE

HocheggerH

TakedaS

2004 Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair (Amst) 3 1175 1185

22. SonodaE

HocheggerH

SaberiA

TaniguchiY

TakedaS

2006 Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) 5 1021 1029

23. Yamaguchi-IwaiY

SonodaE

SasakiMS

MorrisonC

HaraguchiT

1999 Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells. Embo J 18 6619 6629

24. NakaharaM

SonodaE

NojimaK

SaleJE

TakenakaK

2009 Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig v in chicken B lymphocytes. PLoS Genet 5 e1000356 doi:10.1371/journal.pgen.1000356

25. SonodaE

SasakiMS

BuersteddeJM

BezzubovaO

ShinoharaA

1998 Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. Embo J 17 598 608

26. PolanowskaJ

MartinJS

Garcia-MuseT

PetalcorinMI

BoultonSJ

2006 A conserved pathway to activate BRCA1-dependent ubiquitylation at DNA damage sites. Embo J 25 2178 2188

27. ZhaoGY

SonodaE

BarberLJ

OkaH

MurakawaY

2007 A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol Cell 25 663 675

28. FukushimaT

TakataM

MorrisonC

ArakiR

FujimoriA

2001 Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair. J Biol Chem 276 44413 44418

29. TauchiH

KobayashiJ

MorishimaK

van GentDC

ShiraishiT

2002 Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature 420 93 98

30. TakataM

SasakiMS

SonodaE

MorrisonC

HashimotoM

1998 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. Embo J 17 5497 5508

31. MartinRW

OrelliBJ

YamazoeM

MinnAJ

TakedaS

2007 RAD51 up-regulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors. Cancer Res 67 9658 9665

32. JiK

KogameT

ChoiK

WangX

LeeJ

2009 A novel approach to screening and characterizing the genotoxicity of environmental contaminants using DNA-repair-deficient chicken DT40 cell lines. Environ Health Perspect 117 1737 1744

33. ChenPL

LiuF

CaiS

LinX

LiA

2005 Inactivation of CtIP leads to early embryonic lethality mediated by G1 restraint and to tumorigenesis by haploid insufficiency. Mol Cell Biol 25 3535 3542

34. LiuF

LeeWH

2006 CtIP activates its own and cyclin D1 promoters via the E2F/RB pathway during G1/S progression. Mol Cell Biol 26 3124 3134

35. TakaoN

KatoH

MoriR

MorrisonC

SonadaE

1999 Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene 18 7002 7009

36. SwanbergSE

PayneWS

HuntHD

DodgsonJB

DelanyME

2004 Telomerase activity and differential expression of telomerase genes and c-myc in chicken cells in vitro. Dev Dyn 231 14 21

37. YunMH

HiomK

2009 CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle. Nature 459 460 463

38. LimboO

ChahwanC

YamadaY

de BruinRA

WittenbergC

2007 Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination. Mol Cell 28 134 146

39. MaserRS

ZinkelR

PetriniJH

2001 An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele. Nat Genet 27 417 421

40. SchlegelBP

JodelkaFM

NunezR

2006 BRCA1 promotes induction of ssDNA by ionizing radiation. Cancer Res 66 5181 5189

41. BhattacharyyaA

EarUS

KollerBH

WeichselbaumRR

BishopDK

2000 The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin. J Biol Chem 275 23899 23903

42. SySM

HuenMS

ChenJ

2009 PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc Natl Acad Sci U S A 106 7155 7160

43. YangSW

BurginABJr

HuizengaBN

RobertsonCA

YaoKC

1996 A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases. Proc Natl Acad Sci U S A 93 11534 11539

44. CaldecottKW

2008 Single-strand break repair and genetic disease. Nat Rev Genet 9 619 631

45. SordetO

LarochelleS

NicolasE

StevensEV

ZhangC

2008 Hyperphosphorylation of RNA polymerase II in response to topoisomerase I cleavage complexes and its association with transcription- and BRCA1-dependent degradation of topoisomerase I. J Mol Biol 381 540 549

46. OkadaT

SonodaE

YamashitaYM

KoyoshiS

TateishiS

2002 Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage. J Biol Chem 277 48690 48695

47. KikuchiK

TaniguchiY

HatanakaA

SonodaE

HocheggerH

2005 Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends. Mol Cell Biol 25 6948 6955

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