Impactos de las energías en el ecosistema

Coomassie blue staining. Arrows indicate each protein or subunit. The asterisk represents impurities in the purified protein. The numbers to the left of the Coomassie-stained gel indicates the location of the molecular weight markers (kDa). The indicated GST-Claspin fragments shown in Figure 3.2 were mixed either B Pol ε, C Rad17-RFC, or D Timeless, retrieved with glutathione resin, and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The GST-Claspin fragments were visualized by staining the membrane with Ponceau S. Input represents 10% of the protein tested in the binding assays. The relative amount of each protein that co-precipitated with each Claspin fragment was quantified and normalized relative to the input signal. Error bars represent the standard deviation of the mean from two (Timeless) or three independent experiments (Pol ε, Rad17-RFC).

89

Figure 3.4

Claspin directly interacts with Cdc45.

A GST-Cdc45 and B GST-Cdc25C were purified from E. coli with glutathione resin and analyzed by SDS-PAGE and Coomassie blue staining. The arrows indicate the full-length purified proteins. The numbers to the left of the Coomassie-stained gel indicates the location of the molecular weight markers (kDa). The purified GST-Cdc45 had a number of degradation products, as visualized in the Coomassie-stained gel and verified by immunoblotting with an anti-Cdc45 antibody. C Flag-tagged Claspin fragments shown in Figure 2 were mixed with either GST-Cdc45 or GST-Cdc25C, incubated with glutathione resin, and bound proteins analyzed by SDS-PAGE and immunoblotting with anti-Flag antibody and Ponceau S-staining of the membrane. D The relative amount of each protein that co-precipitated with each GST-tagged protein was quantified and normalized relative to the input signal (10% of material used in the binding assay). Error bars represent the standard deviation of the mean from three independent experiments.

90

Figure 3.5

The Claspin N-terminus but not the C-terminus binds to replication fork-like DNA in vitro. Each of indicated the GST-Claspin fragments (20, 40, and 100 nM) was incubated with a biotinylated, branched DNA substrate (20 nM) immobilized on magnetic beads, as described in the Materials and Methods section. Binding reactions were performed with either A 50 mM NaCl or B 100 mM NaCl in the binding and wash buffer. Proteins bound to the DNA- beads were analyzed by SDS-PAGE and immunoblotting. Input lanes contain 0.5 pmol of the indicated protein. Signals were quantified using the input signal for normalization and then graphed. Data represent the mean and standard deviation from three independent experiments.

91

Figure 3.6

Schematic of Claspin functional domains.

A summary of the functional regions of Claspin are indicated, based on data described in the text. BPI and BPII indicate basic patches I and II. RFID, replication fork-interacting domain. CKB, Chk1-binding domain. MRC1, Mrc1-like domain.

92

FIGURES

Figure 3.1

93

Figure 3.2

94

Figure 3.3

Claspin directly interacts with Pol ε, Rad17-RFC, and Timeless.

95

Figure 3.3

96

Figure 3.4

97

Figure 3.5

98

Figure 3.6

99

REFERENCES

1. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004; 73:39-85.

2. Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Mol Cell 2010; 40:179-204.

3. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300:1542-1548.

4. Bermudez VP, Lindsey-Boltz LA, Cesare AJ, Maniwa Y, Griffith JD, Hurwitz J, Sancar A. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci U S A 2003; 100:1633-1638.

5. Kumagai A, Lee J, Yoo HY, Dunphy WG. TopBP1 activates the ATR-ATRIP complex. Cell 2006; 124:943-955.

6. Delacroix S, Wagner JM, Kobayashi M, Yamamoto K, Karnitz LM. The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev 2007; 21:1472-1477. 7. Cimprich KA, Cortez D. ATR: An essential regulator of genome integrity. Nat Rev Mol Cell Biol 2008; 9:616-627.

8. Kumagai A, Dunphy WG. Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in xenopus egg extracts. Mol Cell 2000; 6:839-849.

9. Kumagai A, Kim SM, Dunphy WG. Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1. J Biol Chem 2004; 279:49599-49608.

10. Chini CC, Chen J. Human claspin is required for replication checkpoint control. J Biol Chem 2003; 278:30057-30062.

11. Lin SY, Li K, Stewart GS, Elledge SJ. Human claspin works with BRCA1 to both positively and negatively regulate cell proliferation. Proc Natl Acad Sci U S A 2004; 101:6484-6489.

12. Liu S, Bekker-Jensen S, Mailand N, Lukas C, Bartek J, Lukas J. Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation. Mol Cell Biol 2006; 26:6056-6064.

100

13. Lupardus PJ, Cimprich KA. Phosphorylation of xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of claspin and the Rad9 carboxy terminus. Mol Biol Cell 2006; 17:1559-1569.

14. Lindsey-Boltz LA, Sercin O, Choi JH, Sancar A. Reconstitution of human claspin- mediated phosphorylation of Chk1 by the ATR (ataxia telangiectasia-mutated and rad3- related) checkpoint kinase. J Biol Chem 2009; 284:33107-33114.

15. Alcasabas AA, Osborn AJ, Bachant J, Hu F, Werler PJ, Bousset K, Furuya K, Diffley JF, Carr AM, Elledge SJ. Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat Cell Biol 2001; 3:958-965.

16. Tanaka K, Russell P. Mrc1 channels the DNA replication arrest signal to checkpoint kinase Cds1. Nat Cell Biol 2001; 3:966-972.

17. Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P. Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 2005; 19:699- 706.

18. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 2003; 424:1078-1083.

19. Szyjka SJ, Viggiani CJ, Aparicio OM. Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Mol Cell 2005; 19:691-697.

20. Lou H, Komata M, Katou Y, Guan Z, Reis CC, Budd M, Shirahige K, Campbell JL. Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Mol Cell 2008; 32:106-117.

21. Freudenreich CH, Lahiri M. Structure-forming CAG/CTG repeat sequences are sensitive to breakage in the absence of Mrc1 checkpoint function and S-phase checkpoint signaling: Implications for trinucleotide repeat expansion diseases. Cell Cycle 2004; 3:1370-1374. 22. Razidlo DF, Lahue RS. Mrc1, Tof1 and Csm3 inhibit CAG.CTG repeat instability by at least two mechanisms. DNA Repair (Amst) 2008; 7:633-640.

23. Voineagu I, Narayanan V, Lobachev KS, Mirkin SM. Replication stalling at unstable inverted repeats: Interplay between DNA hairpins and fork stabilizing proteins. Proc Natl Acad Sci U S A 2008; 105:9936-9941.

101

24. Voineagu I, Surka CF, Shishkin AA, Krasilnikova MM, Mirkin SM. Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility. Nat Struct Mol Biol 2009; 16:226-228.

25. Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson RD, Labib K. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol 2006; 8:358-366.

26. Bando M, Katou Y, Komata M, Tanaka H, Itoh T, Sutani T, Shirahige K. Csm3, Tof1, and Mrc1 form a heterotrimeric mediator complex that associates with DNA replication forks. J Biol Chem 2009; 284:34355-34365.

27. Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A, Taskov HB, Shevchenko A, Stewart AF, Stoynov SS. Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol 2005; 347:509-521. 28. Lee J, Gold DA, Shevchenko A, Shevchenko A, Dunphy WG. Roles of replication fork- interacting and Chk1-activating domains from claspin in a DNA replication checkpoint response. Mol Biol Cell 2005; 16:5269-5282.

29. Petermann E, Helleday T, Caldecott KW. Claspin promotes normal replication fork rates in human cells. Mol Biol Cell 2008; 19:2373-2378.

30. Scorah J, McGowan CH. Claspin and Chk1 regulate replication fork stability by different mechanisms. Cell Cycle 2009; 8:1036-1043.

31. Focarelli ML, Soza S, Mannini L, Paulis M, Montecucco A, Musio A. Claspin inhibition leads to fragile site expression. Genes Chromosomes Cancer 2009; 48:1083-1090.

32. Sar F, Lindsey-Boltz LA, Subramanian D, Croteau DL, Hutsell SQ, Griffith JD, Sancar A. Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures. J Biol Chem 2004; 279:39289-39295.

33. Zhao H, Russell P. DNA binding domain in the replication checkpoint protein Mrc1 of schizosaccharomyces pombe. J Biol Chem 2004; 279:53023-53027.

34. Lee J, Kumagai A, Dunphy WG. Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol Cell 2003; 11:329- 340.

35. Tanaka H, Kubota Y, Tsujimura T, Kumano M, Masai H, Takisawa H. Replisome progression complex links DNA replication to sister chromatid cohesion in xenopus egg extracts. Genes Cells 2009; 14:949-963.

102

36. Ilves I, Petojevic T, Pesavento JJ, Botchan MR. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell 2010; 37:247-258.

37. Moyer SE, Lewis PW, Botchan MR. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 2006; 103:10236-10241.

38. Pacek M, Walter JC. A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. EMBO J 2004; 23:3667-3676.

39. Errico A, Costanzo V, Hunt T. Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in xenopus egg extracts. Proc Natl Acad Sci U S A 2007.

40. Gotter AL. A timeless debate: Resolving TIM's noncircadian roles with possible clock function. Neuroreport 2006; 17:1229-1233.

41. Wang X, Zou L, Lu T, Bao S, Hurov KE, Hittelman WN, Elledge SJ, Li L. Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress. Mol Cell 2006; 23:331-341.

42. Gotter AL, Suppa C, Emanuel BS. Mammalian TIMELESS and tipin are evolutionarily conserved replication fork-associated factors. J Mol Biol 2007; 366:36-52.

43. Kemp MG, Akan Z, Yilmaz S, Grillo M, Smith-Roe SL, Kang TH, Cordeiro-Stone M, Kaufmann WK, Abraham RT, Sancar A, Unsal-Kacmaz K. Tipin-RPA interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress. J Biol Chem 2010.

44. Pursell ZF, Isoz I, Lundstrom EB, Johansson E, Kunkel TA. Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 2007; 317:127-130.

45. Lindsey-Boltz LA, Bermudez VP, Hurwitz J, Sancar A. Purification and characterization of human DNA damage checkpoint rad complexes. Proc Natl Acad Sci U S A 2001; 98:11236-11241.

46. Tanaka T, Yokoyama M, Matsumoto S, Fukatsu R, You Z, Masai H. Fission yeast Swi1- Swi3 complex facilitates DNA binding of Mrc1. J Biol Chem 2010; 285:39609-39622. 47. Unsal-Kacmaz K, Mullen TE, Kaufmann WK, Sancar A. Coupling of human circadian and cell cycles by the timeless protein. Mol Cell Biol 2005; 25:3109-3116.

48. Noguchi E, Noguchi C, Du LL, Russell P. Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. Mol Cell Biol 2003; 23:7861-7874.

103

49. Noguchi E, Noguchi C, McDonald WH, Yates JR,3rd, Russell P. Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol Cell Biol 2004; 24:8342-8355.

50. Liu P, Barkley LR, Day T, Bi X, Slater DM, Alexandrow MG, Nasheuer HP, Vaziri C. The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2- independent mechanism. J Biol Chem 2006; 281:30631-30644.

51. Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K. Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev 2005; 19:1905-1919.

52. Unsal-Kacmaz K, Chastain PD, Qu PP, Minoo P, Cordeiro-Stone M, Sancar A, Kaufmann WK. The human Tim/Tipin complex coordinates an intra-S checkpoint response to UV that slows replication fork displacement. Mol Cell Biol 2007; 27:3131-3142.

53. Savitsky PA, Finkel T. Redox regulation of Cdc25C. J Biol Chem 2002; 277:20535- 20540.

54. Chang MS, Sasaki H, Campbell MS, Kraeft SK, Sutherland R, Yang CY, Liu Y, Auclair D, Hao L, Sonoda H, Ferland LH, Chen LB. HRad17 colocalizes with NHP2L1 in the nucleolus and redistributes after UV irradiation. J Biol Chem 1999; 274:36544-36549. 55. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983; 11:1475-1489.

CHAPTER 4: