Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism


The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.


Vyšlo v časopise: Ddc2 Mediates Mec1 Activation through a Ddc1- or Dpb11-Independent Mechanism. PLoS Genet 10(2): e32767. doi:10.1371/journal.pgen.1004136
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004136

Souhrn

The protein kinase Mec1 (ATR ortholog) and its partner Ddc2 (ATRIP ortholog) play a key role in DNA damage checkpoint responses in budding yeast. Previous studies have established the model in which Ddc1, a subunit of the checkpoint clamp, and Dpb11, related to TopBP1, activate Mec1 directly and control DNA damage checkpoint responses at G1 and G2/M. In this study, we show that Ddc2 contributes to Mec1 activation through a Ddc1- or Dpb11-independent mechanism. The catalytic activity of Mec1 increases after DNA damage in a Ddc2-dependent manner. In contrast, Mec1 activation occurs even in the absence of Ddc1 and Dpb11 function at G2/M. Ddc2 recruits Mec1 to sites of DNA damage. To dissect the role of Ddc2 in Mec1 activation, we isolated and characterized a separation-of-function mutation in DDC2, called ddc2-S4. The ddc2-S4 mutation does not affect Mec1 recruitment but diminishes Mec1 activation. Mec1 phosphorylates histone H2A in response to DNA damage. The ddc2-S4 mutation decreases phosphorylation of histone H2A more significantly than the absence of Ddc1 and Dpb11 function does. Our results suggest that Ddc2 plays a critical role in Mec1 activation as well as Mec1 localization at sites of DNA damage.


Zdroje

1. ElledgeSJ (1996) Cell cycle checkpoints: preventing an identity crisis. Science 274: 1664–1672.

2. HarperJW, ElledgeSJ (2007) The DNA damage response: ten years after. Mol Cell 28: 739–745.

3. CimprichKA, CortezD (2008) ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 9: 616–627.

4. HarrisonJC, HaberJE (2006) Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet 40: 209–235.

5. PaciottiV, ClericiM, LucchiniG, LongheseMP (2000) The checkpoint protein Ddc2, functionally related to S. pombe Rad26, interacts with Mec1 and is regulated by Mec1-dependent phosphorylation in budding yeast. Genes & Dev 14: 2046–2059.

6. RouseJ, JacksonSP (2000) LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae. EMBO J 19: 5793–5800.

7. WakayamaT, KondoT, AndoS, MatsumotoK, SugimotoK (2001) Pie1, a protein interacting with Mec1, controls cell growth and checkpoint responses in Saccharomyces cerevisiae. Mol Cell Biol 21: 755–764.

8. RouseJ, JacksonSP (2002) Lcd1p recruits Mec1p to DNA lesions in vitro and in vivo. Mol Cell 9: 857–869.

9. ZouL, ElledgeSJ (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542–1548.

10. NakadaD, HiranoY, SugimotoK (2004) Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway. Mol Cell Biol 24: 10016–10025.

11. NakadaD, HiranoY, TanakaY, SugimotoK (2005) Role of the C terminus of mec1 checkpoint kinase in its localization to sites of DNA damage. Mol Biol Cell 16: 5227–5235.

12. DownsJA, LowndesNF, JacksonSP (2000) A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408: 1001–1004.

13. van AttikumH, GasserSM (2005) The histone code at DNA breaks: a guide to repair? Nat Rev Mol Cell Biol 6: 757–765.

14. GiannattasioM, LazzaroF, PlevaniP, Muzi-FalconiM (2005) The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J Biol Chem 280: 9879–9886.

15. TohGW, O'ShaughnessyAM, JimenoS, DobbieIM, GrenonM, et al. (2006) Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amst) 5: 693–703.

16. EmiliA (1998) MEC1-dependent phosphorylation of Rad9p in response to DNA damage. Mol Cell 2: 183–189.

17. VialardJE, GilbertCS, GreenCM, LowndesNF (1998) The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage. EMBO J 17: 5679–5688.

18. SunZ, HsiaoJ, FayDS, SternDF (1998) Rad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science 281: 272–274.

19. DurocherD, HenckelJ, FershtAR, JacksonSP (1999) The FHA domain is a modular phosphopeptide recognition motif. Mol Cell 4: 387–394.

20. SchwartzMF, DuongJK, SunZ, MorrowJS, PradhanD, et al. (2002) Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Mol Cell 9: 1055–1065.

21. GilbertCS, GreenCM, LowndesNF (2001) Budding yeast Rad9 is an ATP-dependent Rad53 activating machine. Mol Cell 8: 129–136.

22. SmolkaMB, AlbuquerqueCP, ChenSH, SchmidtKH, WeiXX, et al. (2005) Dynamic changes in protein-protein interaction and protein phosphorylation probed with amine-reactive isotope tag. Mol Cell Proteomics 4: 1358–1369.

23. SweeneyFD, YangF, ChiA, ShabanowitzJ, HuntDF, et al. (2005) Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation. Curr Biol 15: 1364–1375.

24. KondoT, MatsumotoK, SugimotoK (1999) Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway. Mol Cell Biol 19: 1136–1143.

25. MajkaJ, BurgersPM (2003) Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint. Proc Natl Acad Sci USA 100: 2249–2254.

26. GreenCM, Erdjument-BromageH, TempstP, LowndesNF (2000) A novel Rad24 checkpoint protein complex closely related to replication factor C. Curr Biol 10: 39–42.

27. NaikiT, ShimomuraT, KondoT, MatsumotoK, SugimotoK (2000) Rfc5, in cooperation with Rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae. Mol Cell Biol 20: 5888–5896.

28. MajkaJ, BinzSK, WoldMS, BurgersPM (2006) Replication protein A directs loading of the DNA damage checkpoint clamp to 5′-DNA junctions. J Biol Chem 281: 27855–27861.

29. KondoT, WakayamaT, NaikiT, MatsumotoK, SugimotoK (2001) Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through distinct mechanisms. Science 5543: 867–870.

30. MeloJA, CohenJ, ToczyskiDP (2001) Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes & Dev 21: 2809–2821.

31. BonillaCY, MeloJA, ToczyskiDP (2008) Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage. Mol Cell 30: 267–276.

32. PudduF, GranataM, Di NolaL, BalestriniA, PiergiovanniG, et al. (2008) Phosphorylation of the budding yeast 9-1-1 complex is required for Dpb11 function in the full activation of the UV-induced DNA damage checkpoint. Mol Cell Biol 28: 4782–4793.

33. LongheseMP, PaciottiV, FraschiniR, ZaccariniR, PlevaniP, et al. (1997) The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast. EMBO J 16: 5216–5226.

34. Navadgi-PatilVM, BurgersPM (2009) The unstructured C-terminal tail of the 9-1-1 clamp subunit Ddc1 activates Mec1/ATR via two distinct mechanisms. Mol Cell 36: 743–753.

35. Navadgi-PatilVM, KumarS, BurgersPM (2011) The unstructured C-terminal tail of yeast Dpb11 (human TopBP1) protein is dispensable for DNA replication and the S phase checkpoint but required for the G2/M checkpoint. J Biol Chem 286: 40999–41007.

36. PudduF, PiergiovanniG, PlevaniP, Muzi-FalconiM (2011) Sensing of replication stress and Mec1 activation act through two independent pathways involving the 9-1-1 complex and DNA polymerase epsilon. PLoS Genet 7: e1002022.

37. MajkaJ, Niedziela-MajkaA, BurgersPM (2006) The checkpoint clamp activates Mec1 kinase during initiation of the DNA damage checkpoint. Mol Cell 24: 891–901.

38. MordesDA, NamEA, CortezD (2008) Dpb11 activates the Mec1-Ddc2 complex. Proc Natl Acad Sci U S A 105: 18730–18734.

39. Navadgi-PatilVM, BurgersPM (2008) Yeast DNA replication protein Dpb11 activates the Mec1/ATR checkpoint kinase. J Biol Chem 283: 35853–35859.

40. PfanderB, DiffleyJF (2011) Dpb11 coordinates Mec1 kinase activation with cell cycle-regulated Rad9 recruitment. EMBO J 30: 4897–4907.

41. KumarS, BurgersPM (2013) Lagging strand maturation factor Dna2 is a component of the replication checkpoint initiation machinery. Genes Dev 27: 313–321.

42. NakadaD, ShimomuraT, MatsumotoK, SugimotoK (2003) The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment. Nucleic Acids Res 31: 1715–1724.

43. ArakiH, LeemSH, PhongdaraA, SuginoA (1995) Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc Natl Acad Sci U S A 92: 11791–11795.

44. WoldMS (1997) Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66: 61–92.

45. CortezD, GuntukuS, QinJ, ElledgeSJ (2001) ATR and ATRIP: partners in checkpoint signaling. Science 294: 1713–1716.

46. KimSM, KumagaiA, LeeJ, DunphyWG (2005) Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP. J Biol Chem 280: 38355–38364.

47. BallHL, MyersJS, CortezD (2005) ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation. Mol Biol Cell 16: 2372–2381.

48. NamikiY, ZouL (2006) ATRIP associates with replication protein A-coated ssDNA through multiple interactions. Proc Natl Acad Sci U S A 103: 580–585.

49. MordesDA, GlickGG, ZhaoR, CortezD (2008) TopBP1 activates ATR through ATRIP and a PIKK regulatory domain. Genes Dev 22: 1478–1489.

50. ItakuraE, TakaiKK, UmedaK, KimuraM, OhsumiM, et al. (2004) Amino-terminal domain of ATRIP contributes to intranuclear relocation of the ATR-ATRIP complex following DNA damage. FEBS Lett 577: 289–293.

51. BallHL, CortezD (2005) ATRIP oligomerization is required for ATR-dependent checkpoint signaling. J Biol Chem 280: 31390–31396.

52. BallHL, EhrhardtMR, MordesDA, GlickGG, ChazinWJ, et al. (2007) Function of a conserved checkpoint recruitment domain in ATRIP proteins. Mol Cell Biol 27: 3367–3377.

53. NaikiT, WakayamaT, NakadaD, MatsumotoK, SugimotoK (2004) Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism. Mol Cell Biol 24: 3277–3285.

54. GranataM, LazzaroF, NovarinaD, PanigadaD, PudduF, et al. (2010) Dynamics of Rad9 chromatin binding and checkpoint function are mediated by its dimerization and are cell cycle-regulated by CDK1 activity. PLoS Genet 6: e1001047.

55. LiuS, ShiotaniB, LahiriM, MarechalA, TseA, et al. (2011) ATR autophosphorylation as a molecular switch for checkpoint activation. Mol Cell 43: 192–202.

56. BerensTJ, ToczyskiDP (2012) Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo. Mol Biol Cell 23: 1058–1067.

57. JamesP, HalladayJ, CraigEA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425–1436.

58. GietzRD, SuginoA (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74: 527–534.

59. ReidRJ, LisbyM, RothsteinR (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350: 258–277.

60. WachA, BrachatA, PohlmannR, PhilippsenP (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 13: 1793–1808.

61. FukunagaK, KwonY, SungP, SugimotoK (2011) Activation of Protein Kinase Tel1 through Recognition of Protein-Bound DNA Ends. Mol Cell Biol 31: 1959–1971.

62. BoekeJD, TrueheartJ, NatsoulisG, FinkGR (1987) 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154: 164–175.

63. FukunagaK, HiranoY, SugimotoK (2012) Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast. Mol Biol Cell 23: 347–359.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 2
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Eozinofilní granulomatóza s polyangiitidou
nový kurz
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa