Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1
2009; Elsevier BV; Volume: 284; Issue: 28 Linguagem: Inglês
10.1074/jbc.m109.018242
ISSN1083-351X
Autores Tópico(s)Microtubule and mitosis dynamics
ResumoUpon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint. Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint. Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1.Samadashwily G.M. Raca G. Mirkin S.M. Nat. Genet. 1997; 17: 298-304Crossref PubMed Scopus (288) Google Scholar, 2.Nyberg K.A. Michelson R.J. Putnam C.W. Weinert T.A. Annu. Rev. Genet. 2002; 36: 617-656Crossref PubMed Scopus (653) Google Scholar, 3.Kolodner R.D. Putnam C.D. Myung K. Science. 2002; 297: 552-557Crossref PubMed Scopus (405) Google Scholar). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4.Lopes M. Cotta-Ramusino C. Pellicioli A. Liberi G. Plevani P. Muzi-Falconi M. Newlon C.S. Foiani M. Nature. 2001; 412: 557-561Crossref PubMed Scopus (628) Google Scholar, 5.Santocanale C. Diffley J.F. Nature. 1998; 395: 615-618Crossref PubMed Scopus (539) Google Scholar, 6.Tercero J.A. Diffley J.F. Nature. 2001; 412: 553-557Crossref PubMed Scopus (564) Google Scholar, 7.Desany B.A. Alcasabas A.A. Bachant J.B. Elledge S.J. Genes Dev. 1998; 12: 2956-2970Crossref PubMed Scopus (384) Google Scholar). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8.Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 9.Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2334) Google Scholar). Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR, 2The abbreviations used are: ATRataxia telangiectasia-mutated and Rad3-relatedDTTdithiothreitolNi-NTAnickel-nitrilotriacetic acidWTwild typeRDSRad53-Dun1-Sml1Rad53KDRad53 kinase-dead proteinHUhydroxyureaGSTglutathione S-transferaseFHAforkhead associated. is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10.Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2115) Google Scholar, 11.Nakada D. Hirano Y. Tanaka Y. Sugimoto K. Mol. Biol. Cell. 2005; 16: 5227-5235Crossref PubMed Scopus (45) Google Scholar). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single- and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12.Majka J. Burgers P.M. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2249-2254Crossref PubMed Scopus (221) Google Scholar). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13.Majka J. Niedziela-Majka A. Burgers P.M. Mol. Cell. 2006; 24: 891-901Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14.Garcia V. Furuya K. Carr A.M. DNA Repair. 2005; 4: 1227-1239Crossref PubMed Scopus (149) Google Scholar). In addition to regulating the initiation of DNA replication, they were found to play a role in the DNA replication checkpoint (15.Araki H. Leem S.H. Phongdara A. Sugino A. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 11791-11795Crossref PubMed Scopus (244) Google Scholar, 16.Kamimura Y. Masumoto H. Sugino A. Araki H. Mol. Cell. Biol. 1998; 18: 6102-6109Crossref PubMed Scopus (140) Google Scholar, 17.Wang H. Elledge S.J. Genetics. 2002; 160: 1295-1304Crossref PubMed Google Scholar). They interact with the 9-1-1 complex and directly stimulate Mec1/ATR activity in vitro (18.Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 19.Mordes D.A. Nam E.A. Cortez D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18730-18734Crossref PubMed Scopus (96) Google Scholar, 20.Navadgi-Patil V.M. Burgers P.M. J. Biol. Chem. 2008; 283: 35853-35859Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, the assembly of multiple protein complexes at stalled DNA replication forks appears to facilitate activation of the DNA replication checkpoint (13.Majka J. Niedziela-Majka A. Burgers P.M. Mol. Cell. 2006; 24: 891-901Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 18.Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). ataxia telangiectasia-mutated and Rad3-related dithiothreitol nickel-nitrilotriacetic acid wild type Rad53-Dun1-Sml1 Rad53 kinase-dead protein hydroxyurea glutathione S-transferase forkhead associated. Mrc1 (for mediator of replication checkpoint) was originally identified to be important for cells to respond to hydroxyurea in S. cerevisiae and Schizosaccharomyces pombe (21.Alcasabas A.A. Osborn A.J. Bachant J. Hu F. Werler P.J. Bousset K. Furuya K. Diffley J.F. Carr A.M. Elledge S.J. Nat. Cell Biol. 2001; 3: 958-965Crossref PubMed Scopus (426) Google Scholar, 22.Tanaka K. Russell P. Nat. Cell Biol. 2001; 3: 966-972Crossref PubMed Scopus (202) Google Scholar). Mrc1 is a component of the DNA replisome and travels with the replication forks along chromosome during DNA synthesis (23.Szyjka S.J. Viggiani C.J. Aparicio O.M. Mol. Cell. 2005; 19: 691-697Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 24.Gambus A. Jones R.C. Sanchez-Diaz A. Kanemaki M. van Deursen F. Edmondson R.D. Labib K. Nat. Cell Biol. 2006; 8: 358-366Crossref PubMed Scopus (611) Google Scholar, 25.Lou H. Komata M. Katou Y. Guan Z. Reis C.C. Budd M. Shirahige K. Campbell J.L. Mol. Cell. 2008; 32: 106-117Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Deletion of MRC1 causes defects in DNA replication, indicating its role in the normal progression of DNA replication (23.Szyjka S.J. Viggiani C.J. Aparicio O.M. Mol. Cell. 2005; 19: 691-697Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Interestingly, when DNA replication is blocked by hydroxyurea, Mrc1 undergoes Mec1- and Rad3 (S. pombe ortholog of Mec1)-dependent phosphorylation (21.Alcasabas A.A. Osborn A.J. Bachant J. Hu F. Werler P.J. Bousset K. Furuya K. Diffley J.F. Carr A.M. Elledge S.J. Nat. Cell Biol. 2001; 3: 958-965Crossref PubMed Scopus (426) Google Scholar, 22.Tanaka K. Russell P. Nat. Cell Biol. 2001; 3: 966-972Crossref PubMed Scopus (202) Google Scholar). In S. cerevisiae, mutations of Mrc1 at the (S/T)Q sites, which are consensus phosphorylation sites of the Mec1/ATR family kinases, abolishes hydroxyurea-induced Mrc1 phosphorylation in vivo, suggesting a direct phosphorylation of Mrc1 by Mec1 (21.Alcasabas A.A. Osborn A.J. Bachant J. Hu F. Werler P.J. Bousset K. Furuya K. Diffley J.F. Carr A.M. Elledge S.J. Nat. Cell Biol. 2001; 3: 958-965Crossref PubMed Scopus (426) Google Scholar, 22.Tanaka K. Russell P. Nat. Cell Biol. 2001; 3: 966-972Crossref PubMed Scopus (202) Google Scholar). Rad53 and Cds1, homologs of human Chk2, are the major effector kinases in the DNA replication checkpoints in S. cerevisiae and S. pombe, respectively. Activation of Rad53 is a hallmark of DNA replication checkpoint activation and is important for the maintenance of DNA replication forks in response to DNA replication stress (5.Santocanale C. Diffley J.F. Nature. 1998; 395: 615-618Crossref PubMed Scopus (539) Google Scholar, 6.Tercero J.A. Diffley J.F. Nature. 2001; 412: 553-557Crossref PubMed Scopus (564) Google Scholar). Thus, it is important to understand how Rad53 activity is controlled. Interestingly, mutation of all the (S/T)Q sites of Mrc1 not only abolishes the phosphorylation of Mrc1 by Mec1 but also compromises hydroxyurea-induced Rad53 activation in S. cerevisiae (21.Alcasabas A.A. Osborn A.J. Bachant J. Hu F. Werler P.J. Bousset K. Furuya K. Diffley J.F. Carr A.M. Elledge S.J. Nat. Cell Biol. 2001; 3: 958-965Crossref PubMed Scopus (426) Google Scholar). Similarly, mutation of the TQ sites of Mrc1 in S. pombe was shown to abolish the binding between Cds1 and Mrc1 as well as Cds1 activation (22.Tanaka K. Russell P. Nat. Cell Biol. 2001; 3: 966-972Crossref PubMed Scopus (202) Google Scholar). Further, mutation of specific TQ sites of Mrc1 in S. pombe abolishes its binding to Cds1 in vitro and the activation of Cds1 in vivo (26.Xu Y.J. Davenport M. Kelly T.J. Genes Dev. 2006; 20: 990-1003Crossref PubMed Scopus (72) Google Scholar). Thus, Mec1/Rad3-dependent phosphorylation of Mrc1 is responsible for Mrc1 binding to Rad53/Cds1, which is essential for Rad53/Cds1 activation. An intriguing property of the Chk2 family kinases is their ability to undergo autophosphorylation and activation in the absence of other proteins in vitro (27.Gilbert C.S. Green C.M. Lowndes N.F. Mol. Cell. 2001; 8: 129-136Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 28.Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (162) Google Scholar). First, autophosphorylation of a conserved threonine residue in the activation loop of Chk2 family kinase was found to be an essential part of their activation processes (26.Xu Y.J. Davenport M. Kelly T.J. Genes Dev. 2006; 20: 990-1003Crossref PubMed Scopus (72) Google Scholar, 29.Chen S.H. Smolka M.B. Zhou H. J. Biol. Chem. 2007; 282: 986-995Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30.Ahn J.Y. Li X. Davis H.L. Canman C.E. J. Biol. Chem. 2002; 277: 19389-19395Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 31.Usui T. Petrini J.H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2797-2802Crossref PubMed Scopus (33) Google Scholar). Second, a direct and trans-phosphorylation of the N-terminal TQ sites of the Chk2 family kinases by the Mec1/ATR family kinases is also important for their activation in vivo. Analogous to the requirement of N-terminal TQ site phosphorylation of Chk2 by ATR in human (32.Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10389-10394Crossref PubMed Scopus (701) Google Scholar), the activation of Rad53/Cds1 in vivo requires phosphorylation of TQ sites in their N termini by Mec1/Rad3 (33.Lee S.J. Schwartz M.F. Duong J.K. Stern D.F. Mol. Cell. Biol. 2003; 23: 6300-6314Crossref PubMed Scopus (90) Google Scholar, 34.Tanaka K. Boddy M.N. Chen X.B. McGowan C.H. Russell P. Mol. Cell. Biol. 2001; 21: 3398-3404Crossref PubMed Scopus (48) Google Scholar). Considering that Mec1, Mrc1, and many other proteins are recruited at stalled DNA replication forks and have been shown to be involved in DNA replication checkpoint activation, a key question remains unresolved: what is the minimal system that is capable of activating Rad53 directly? Given the direct physical interaction between Mrc1 and Rad53 and the requirement of Mrc1 and Mec1 in vivo, it is likely that they both play a role in Rad53 activation. Furthermore, what is the molecular mechanism of Rad53 activation by its upstream activators? To address these questions, a faithful reconstitution of the activation of Rad53 using purified proteins is necessary. In this study, we developed an activity-based assay consisting of the Dun1 kinase, a downstream substrate of Rad53, and Sml1, as a substrate of Dun1, to quantitatively measure the activity of Rad53. Using this coupled kinase assay from Rad53 to Dun1 and then to Sml1, we screened for Mrc1 and its associated factors to see whether they could directly activate Rad53 in vitro. Our results showed that Mec1 and Mrc1 collaborate to constitute a minimal system in direct activation of Rad53. The plasmids used are summarized in supplemental Table S1. Yeast strains are summarized in supplemental Table S2. MRC1 was first cloned into the pFA6a-3XHA-Kan plasmid using PacI and AscI followed by mutagenesis. Various mutations of MRC1 were then introduced into yeast cells via homologous recombination (35.Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4281) Google Scholar). All plasmids and mutations introduced into yeast cells were confirmed by DNA sequencing. All purification steps were performed at 4 °C. 2 liters of yeast cells (SCY216, SCY152, and SCY230 for mec1Δ, rad53Δ, and Mrc1-TAF/rad53Δ, respectively) were grown in YPD (yeast extract, peptone, and dextrose) medium to log phase (A600 ∼ 0.8). Spheroplasts were prepared as described (36.Pasero P. Duncker B.P. Gasser S.M. Methods. 1999; 18: 368-376, 323Crossref PubMed Scopus (6) Google Scholar). The extract was prepared from spheroplasts in 10 ml of buffer A (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm DTT, 0.1% Tween 20, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1.5 μm pepstatin A, 1 μm leupeptin, 0.2 mm benzamidine, 5 mm β-glycerophosphate, 5 mm sodium fluoride) by sonication and clarification at 70,000 rpm in a MLA80 rotor for 10 min. 200 μl of anti-FLAG M2 affinity resin (Sigma) was added to 10 ml of the extract (∼30 μg/μl protein), and the bead/extract mix was rotated for 3 h. The anti-FLAG resins were then washed with 15 bead volumes of TBSD (50 mm Tris-HCl, pH 7.5, 150 mm sodium chloride, 1 mm DTT, 0.1% Tween 20), and proteins were eluted by incubation with 1 bead volume of TBSD containing 10% glycerol and 200 μg/ml 3× FLAG peptide (Sigma) for 1 h at room temperature. Elution equivalent to 0.5 liter of yeast culture was prepared for mass spectrometry analysis as follows. Elution was denatured with 1% SDS followed by reduction and alkylation with 10 mm DTT and 30 mm iodoacetamide, respectively. Proteins were then precipitated and washed with 50% ethanol, 50% acetone, and 0.1% acetic acid. The precipitated proteins were then resuspended and subjected to trypsin (Roche Applied Science) digestion. Mass spectrometry analysis was similar as described previously (37.Smolka M.B. Chen S.H. Maddox P.S. Enserink J.M. Albuquerque C.P. Wei X.X. Desai A. Kolodner R.D. Zhou H. J. Cell Biol. 2006; 175: 743-753Crossref PubMed Scopus (68) Google Scholar) except a Thermo Finnigan (Thermo Scientific, Waltham, MA) LTQ mass spectrometer was used for sample analysis. 2 liters of yeast cells were grown in YPD medium to log phase and broken in an ice-cooled bead beater (Hamilton Beach/Proctor-Silex, Inc.) in 40 ml of buffer A. Crude extracts were clarified by centrifugation at 15,000 rpm in a JA-25.50 rotor for 30 min and added to ∼100 μl of anti-FLAG M2 resins and IgG resins (IgG-Sepharose 6 Fast Flow, GE Healthcare) for the immunoprecipitation of Rad53 and Dun1, respectively. The bead/extract mix was rotated for 3 h, and the resins were washed with 5 bead volumes of TBSD supplemented with 1 m sodium chloride followed by washing with 15 bead volumes of TBSD (standard wash). Rad53 was eluted by 2 bead volumes of TBSD containing 200 μg/ml 3× FLAG peptide and then bound to 20 μl of Ni-NTA resin (Qiagen) for 2 h. The Ni-NTA resins were washed, and Rad53 was eluted by 4 bead volumes of TBSD containing 10% glycerol and 200 mm imidazole. Dun1 was eluted by incubation with 2 bead volumes TBSD containing 10 unit of tobacco etch virus protease for 2 h at 30 °C, and the supernatant was added to 20 μl of anti-FLAG M2 resin for 2 h. The anti-FLAG M2 column was washed, and Dun1 was eluted by incubation with 4 bead volumes of TBSD containing 10% glycerol and 200 μg/ml 3× FLAG peptide for 1 h at room temperature. Both Rad53 and Dun1 purifications yielded a protein concentration of ∼20 ng/μl. Concentrations of Rad53 and Dun1 were determined by comparison with a known bovine serum albumin standard using 10% SDS-PAGE analysis and silver staining. BL21 cells were used to overexpress Rad53 and Rad53 kinase-dead protein (Rad53KD) using plasmids HZE1452 and HZE1446, respectively. Extract was prepared from 2 liters of cells in 20 ml of buffer A by sonication and clarification at 30,000 rpm in a JA-25.50 rotor for 30 min. 250 μl of IgG resins were added to the extract, the bead/extract mix was rotated for 2 h, and the IgG resins were washed. For the purification of active Rad53, Rad53 was eluted using 3 bead volumes of TBSD containing 10 units of PreScission protease (Amersham Biosciences); and the supernatant was bound to 100 μl of Ni-NTA resins for 2 h. The Ni-NTA resins were washed, and Rad53 was eluted by 2 bead volumes of TBSD containing 200 mm imidazole. Eluted Rad53 was dialyzed in TBSD supplemented with 10% glycerol. The final Rad53 concentration was 500 ng/μl. For the purification of inactive Rad53, all the steps were the same except for an additional inaction step after the IgG binding of Rad53. To inactive/dephosphorylate Rad53, IgG-bound Rad53 was incubated with 1000 units of Lambda phosphatase (New England BioLabs) in 1 bead volume of TBSD supplemented with 5 mm magnesium chloride and 2% glycerol for 12 h at room temperature. Following the wash to remove Lambda phosphatase, Rad53 was eluted by PreScission protease. BL21 cells were used to overexpress Mrc1 WT and various mutant proteins using the plasmids HZE1516-1521. The purification of these recombinant proteins was similar to that described above for Rad53. The introduction of a His6 tag at the C terminus of Mrc1 incidentally introduced a point mutation in the tag, resulting in a sequence of GGGSSSSSS in the C termini of all Mrc1 proteins used. All of these Mrc1 proteins were still purified using the Ni-NTA resin. Concentration of Mrc1 was determined by comparing with a known bovine serum albumin standard using 10% SDS-PAGE analysis and Coomassie staining. pYES-PP-Dun1 was transformed in SCY152. 100 ml of cells were grown in CSM-Ura 2% glucose to an A600 of 1.5. Cells were pelleted, resuspended in 100 ml of medium containing CSM-Ura, 2% galactose, and 0.1% glucose, and grown for 12 h. An extract was prepared by breaking the cells in 10 ml of buffer A using a vortex (Vortex-Genie 2, Scientific Industries), and centrifugation at 13,200 rpm in a F45-24-11 rotor for 10 min. 100 μl of IgG resins were added to the extract, and the bead/extract mix was rotated for 2 h. After the washing of IgG resins, Dun1 was eluted by incubating it with 2 bead volumes of TBSD containing 10% glycerol and 5 units of PreScission protease overnight. The concentration of purified Dun1 was 500 ng/μl. S. cerevisiae strain SCY001, transformed with plasmid pBL504 (Mec1/GST-Ddc2), was grown and induced with galactose under the same conditions as described above. 12 liters of cells were broken in an ice-cooled bead beater in 300 ml of buffer B (buffer A plus 0.01% Nonidet P-40) and clarified by centrifugation at 15,000 rpm in a JA-25.50 rotor for 30 min. Proteins were precipitated using ammonium sulfate to 55% saturation. The precipitate was collected after centrifugation at 15,000 rpm for 30 min. The protein pellet was resuspended in 30 ml of buffer B and then incubated with 3 ml of anti-FLAG M2 resin for 4 h. After washing, proteins were eluted with 3 bead volumes of TBSD containing 100 μg/ml 3× FLAG peptide. 300 μl of glutathione-Sepharose 4 Fast Flow resin (Amersham Biosciences) was added to the FLAG-eluted sample, and the sample was rotated overnight. The glutathione resins were washed, and Mec1-Ddc2 was eluted by incubating it with 2 bead volumes of TBSD containing 50 units of PreScission protease for 4 h. The eluted sample was loaded onto a 1-ml heparin-agarose column (GE Healthcare), washed with 10 ml of TBSD, and eluted with TBSD containing 500 mm sodium chloride. Finally, eluted Mec1-Ddc2 was dialyzed in TBSD supplemented with 10% glycerol. The final Mec1-Ddc2 concentration was 100 ng/μl. In a typical kinase assay, 20 mm Tris, pH 7.5, 50 mm NaCl, 0.2 mm ATP, 1 mm MgCl2, 1 mm DTT, and 10 μCi of [γ32P]ATP were used. A standard kinase reaction for 30 min at 30 °C was used unless stated otherwise. The binding assay was similar to that described previously (37.Smolka M.B. Chen S.H. Maddox P.S. Enserink J.M. Albuquerque C.P. Wei X.X. Desai A. Kolodner R.D. Zhou H. J. Cell Biol. 2006; 175: 743-753Crossref PubMed Scopus (68) Google Scholar). Briefly, GST fusion proteins of the wild type and R70A mutant FHA1 domains of Rad53 were first purified using glutathione resins (Promega). The GST-FHA1 domain bound glutathione resins were then incubated with either unphosphorylated or phosphorylated recombinant Mrc1-FLAG for 3 h (recombinant Mrc1-FLAG was purified using the same protocol as described for the purification of recombinant Mrc1, except anti-FLAG resins were used during the second step of purification). After washing, the FHA1-bound proteins were eluted by boiling with SDS sample buffer containing 10 mm DTT and then analyzed by 10% SDS-PAGE. This was followed by an anti-FLAG Western blot to detect the presence of Mrc1-FLAG. To phosphorylate Mrc1-Flag by Mec1, 100 μl of kinase reaction containing 80 nm recombinant Mrc1-FLAG with or without 0.6 nm Mec1 was incubated for 2 h at 30 °C. After the kinase reaction, the reaction mixture was incubated with the WT and R70A mutant GST-FHA1 domains of Rad53 (bound to glutathione resins) as described above. To identify factors that may activate Rad53 directly, we developed a Rad53 activity-based assay consisting of inactive Rad53, inactive Dun1, and recombinant Sml1 (Fig. 1, A and 1B). As shown previously (29.Chen S.H. Smolka M.B. Zhou H. J. Biol. Chem. 2007; 282: 986-995Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), Dun1 activation requires Rad53 phosphorylation, and only activated Dun1 can hyperphosphorylate Sml1. We therefore used this inactive Rad53-Dun1-Sml1 (RDS) system as a reporter to identify potential activator(s) of Rad53 (Fig. 1A). As discussed above, Mrc1 is known to bind to Rad53 after its phosphorylation by Mec1 (21.Alcasabas A.A. Osborn A.J. Bachant J. Hu F. Werler P.J. Bousset K. Furuya K. Diffley J.F. Carr A.M. Elledge S.J. Nat. Cell Biol. 2001; 3: 958-965Crossref PubMed Scopus (426) Google Scholar, 37.Smolka M.B. Chen S.H. Maddox P.S. Enserink J.M. Albuquerque C.P. Wei X.X. Desai A. Kolodner R.D. Zhou H. J. Cell Biol. 2006; 175: 743-753Crossref PubMed Scopus (68) Google Scholar). To address whether Mrc1 and its associated proteins could directly activate Rad53 in vitro, we immunoprecipitated epitope-tagged Mrc1-TAF in rad53Δ cells using immobilized anti-FLAG antibody. As controls, parallel anti-FLAG immunoprecipitation experiments were performed using cell extracts from non-epitope-tagged rad53Δ cells and mec1Δ cells. Each immunoprecipitate was then added to the inactive RDS system to perform a kinase reaction with [γ32P]ATP (Fig. 1, A and C). The amount of hyperphosphorylated Sml1 was quantified using scintillation counting. As shown in Fig. 1C, the highest amount of Sml1 phosphorylation was observed using the immunoprecipitated sample from Mrc1-TAF in rad53Δ cells (lane 8). The amount of Sml1 phosphorylation was reduced 10-fold to the basal level when inactive Rad53 was omitted in the kinase assay, indicating that Rad53 is required for Sml1 phosphorylation (see Fig. 1C, lane 5). In contrast, the anti-FLAG-immunoprecipitated sample from either mec1Δ cells or rad53Δ cells was less potent, showing a 2- or 5-fold increase in Sml1 phosphorylation compared with the basal level, respectively. Reproducible results were obtained in repeated experiments leading to the following conclusions. First, Mrc1 facilitates Rad53 activation (Fig. 1C, compare lanes 7 and 8). Second, there are unknown factors that co-purify with Mrc1 to help Rad53 activation (Fig. 1C, compare lane 6 with lanes 7 and 8). Third, these unknown factors are Mec1-dependent and Rad53-independent (Fig. 1C, compare lanes 6 and 7) despite the fact that no epitope-tagged gene was present in mec1Δ and rad53Δ cells. To identify these unknown factors, the immunoprecipitated samples from these cells were analyzed using silver staining (Fig. 1D). Although most of the protein bands are common to all three samples, a distinct band with a molecular weight of more than 250 kDa is present only in lanes 2 and 3 and is absent in lane 1, where mec1Δ cells is used (Fig. 1D). This band was excised from the gel and identified as Mec1 by mass spectrometry. To identify additional specific proteins in these immunoprecipitated samples that were not visualized by silver staining, we performed in-solution trypsin digestion and mass spectrometry analysis. Again, Mec1 and Ddc2 were identified in both samples immunoprecipitated from rad53Δ and Mrc1-TAF/rad53Δ cells but not from mec1Δ cells. In addition, Mrc1 was found only in the sample immunoprecipitated from Mrc1-TAF/rad53Δ cells. These results suggest that Mec1 and Mrc1 may be sufficient and act together to promote Rad53 activation (Fig. 1C), whereas the lack of either Mec1 in the mec1Δ sample or Mrc1 in the rad53Δ sample compromises the activation of Rad53 and thus the hyperphosphorylation of Sml1. The above observation that Mec1 and Mrc1 might act together to activate Rad53 prompted us to examine their effects further. To this end, it is necessary to have sufficient amounts of inactive Rad53 and inactive Dun1. We chose to purify recombinant Rad53 from Escherichia coli, which is known to be active (29.Chen S.H. Smolka M.B. Zhou H. J. Biol. Chem. 2007; 282: 986-995Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). As shown in Fig. 2A, following an extended Lambda phosphatase treatment, recombinant Rad53 is completely dephosphorylated and shows a faster migration in the gel (Fig. 2A). This Lambda phosphatase-dephosphorylated Rad53 has an activity similar to the endogenous Rad53 purified from rad9Δ mrc1Δ cells. Longer Lambda phosphatase treatment had no additional effect on its activity. Thus, it is considered inactive. 3S. Chen and H. Zhou, unpublished observations. To prepare inactive Dun1, Dun1 was overexpressed and purified from rad53Δ cells (Fig. 2A). The activities of these purified kinases were then analyzed using Sml1 as a substrate. Only when active Rad53 and inactive Dun1 and Sml1 were used was a characteristic gel shift of the hyperphosphorylated Sml1 observed, whereas the same amount of inactive Rad53 did not cause appreciable hyperphosphorylation of Sml1 (Fig. 2B). Further characterization of the activities of active Rad53 with inactive Dun1 and Sml1 was carried out to determine the concentration ranges of active Rad53 to be used so that changes in its activity could be better detected (see supplemental Fig. S1). To quantify the difference in activity between active and inactive Rad53, increasing amounts of Rad53 were used to produce Dun1-dependent Sml1 hyperphosphorylation using either active or inactive Rad53. As shown in Fig. 2C, from left to right there is a 2-fold increase in the amount of active or inactive Rad53 in adjacent lanes. Quantification of the hyperphosphorylated Sml1 reveals an ∼250-fold difference in the concentration between active and inactive Rad53 to achieve the same level of Sml1 hyperphosphorylation (Fig. 2D). This allowed us to choose a concentration of inactive Rad53, i.e. 0.5
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