RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule
2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês
10.1074/jbc.m709890200
ISSN1083-351X
AutoresTadashi Nishihara, Fumikiyo Nagawa, Takeshi Imai, Hitoshi Sakano,
Tópico(s)Fungal and yeast genetics research
ResumoIn V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, recombination-activating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule. In V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, recombination-activating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule. 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Our results indicate that the RAG-7-mer interaction is a crucial checkpoint in V(D)J recombination to ensure the 12/23 rule. Preparation of Proteins–The glutathione S-transferase (GST)-tagged truncated RAG1 protein (amino acids 384–1040) was coexpressed with the GST-tagged truncated RAG2 protein (amino acids 1–383) in HEK-293T cells (42Tsai C.L. Schatz D.G. EMBO J. 2003; 22: 1922-1930Crossref PubMed Scopus (63) Google Scholar), purified with glutathione-agarose affinity chromatography (9Spanopoulou E. Zaitseva F. Wang F.H. Santagata S. Baltimore D. Panayotou G. Cell. 1996; 87: 263-276Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), and dialyzed against 25 mm Tris-HCl (pH 8.0), 2 mm dithiothreitol, 150 mm KCl, and 10% glycerol. Plasmids for the RAG1 mutants (HA1, HA2, HA3, and DH3u) were generated by in vitro mutagenesis. DNA Substrates–Oligonucleotides were synthesized and purified as described (43Nagawa F. Hirose S. Nishizumi H. Nishihara T. Sakano H. J. Biol. 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Sequences used were as follows (the 7-mer and 9-mer signal sequences are underlined): 12-RSS top strand (69-mer) for RSS cleavage assay, 5′-CCTGCGCTGAATTCGTCTTACACAGTGCTCCAGGGCTGAACAAAAACCTCCTAGGGTTGCAGCTGACTC-3′; 23-RSS top strand (80-mer) for RSS cleavage assay, 5′-CCTCCTGGGAATTCGTCTTACACAGTGGTAGTACTCCACTGTCTGGGTGTACAAAAACCTCCTAGGGTTGCCATGGACTC-3′; 12-RSS top strand (89-mer) for chemical footprinting, 5′-CTTCAAACCATCCAATAAACCCTGCGCTGAATTCGTCTTACACAGTGCTCCAGGGCTGAACAAAAACCTCCTAGGGTTGCAGCTGACTC-3′; 23-RSS top strand (100-mer) for chemical footprinting, 5′-CTTCAAACCATCCAATAAACCCTGCGCTGAATTCGTCTTACACAGTGGTAGTACTCCACTGTCTGGGTGTACAAAAACCTCCTAGGGTTGCCATGGACTC-3′; the top strand of double-stranded competitor DNA (88-mer) unrelated to the RSS sequence, 5′-CTTCAAACCATCCAATAAACCCTGCGCTGAATTCGTCTTAGCTGAACCTCCAGGGCTGACACCCCCAATCCTAGGGTTGCAGCTGACT-3′. The 32P-labeled, biotinylated, and 3′-dideoxyoligonucleotides were prepared as described (43Nagawa F. Hirose S. Nishizumi H. Nishihara T. Sakano H. J. Biol. Chem. 2004; 279: 38360-38368Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 44Nishihara T. Nagawa F. Nishizumi H. Kodama M. Hirose S. Hayashi R. Sakano H. Mol. Cell Biol. 2004; 24: 3692-3702Crossref PubMed Scopus (8) Google Scholar, 45Nagawa F. Kodama M. Nishihara T. Ishiguro K. Sakano H. Mol. Cell Biol. 2002; 22: 7217-7225Crossref PubMed Scopus (15) Google Scholar). The nicked RSS was prepared by annealing three oligonucleotides: an RSS bottom strand, a 5′-phosphorylated top strand of the signal end DNA, and a coding top strand (with a 3′-dideoxy or 3′-OH end). Annealed DNA was purified by electrophoresis in an 8% polyacrylamide gel, as described (43Nagawa F. Hirose S. Nishizumi H. Nishihara T. Sakano H. J. Biol. Chem. 2004; 279: 38360-38368Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 44Nishihara T. Nagawa F. Nishizumi H. Kodama M. Hirose S. Hayashi R. Sakano H. Mol. Cell Biol. 2004; 24: 3692-3702Crossref PubMed Scopus (8) Google Scholar, 45Nagawa F. Kodama M. Nishihara T. Ishiguro K. Sakano H. Mol. Cell Biol. 2002; 22: 7217-7225Crossref PubMed Scopus (15) Google Scholar). RSS Cleavage Reactions–The 32P-labeled 12-RSS DNA (400 cpm/μl) and the partner RSS DNA (8 nm) were incubated with RAG1 (12 μg/ml), RAG2 (12 μg/ml), and HMG1 (8 μg/ml) proteins at 37 °C for 45 min in cleavage buffer (25 mm MOPS-KOH (pH 7.0), 10 mm Tris-HCl (pH 8.0), 2.4 mm dithiothreitol, 60 mm potassium acetate, 60 mm KCl, 0.1 mg/ml bovine serum albumin, and 2% glycerol) containing 10 mm MgCl2. DNA was extracted with phenol/chloroform/isoamylalchohol (25:24:1), precipitated with ethanol, washed with 70% ethanol, dissolved in formamide dye mix, and electrophoresed in a 10% denaturing polyacrylamide gel. Isolation of RAG-RSS Complexes–To isolate single RSS complex and synaptic complex in parallel, the 32P-labeled nicked RSS DNA (3000 cpm/μl) was incubated with RAG1 D600A (30 μg/ml), RAG2 (30 μg/ml), and HMG1 (8 μg/ml) proteins at 37 °C for 10 min in cleavage buffer containing 0.3 μm competitor DNA and 10 mm MgCl2. The reaction mixture was further incubated at 37 °C for 20 min with or without the nicked partner RSS (16 nm). For the bead separation of the complex, glutathione-Sepharose beads in GST wash buffer (10 mm Tris-HCl (pH 7.4), 120 mm NaCl, 0.1 mg/ml bovine serum albumin, 1 mm dithiothreitol, and 2% glycerol) with 10 mm MgCl2 was added to the reaction mixture. The sample was incubated at 4 °C for 30 min and centrifuged to separate the Sepharose beads. The beads were washed three times with 30 μl of GST wash buffer containing 10 mm MgCl2 and resuspended in 30 μl of binding buffer (25 mm MOPS-KOH (pH 7.0), 10 mm Tris-HCl (pH 8.0), 2.4 mm dithiothreitol, 90 mm potassium acetate, 30 mm KCl, 0.1 mg/ml bovine serum albumin, and 2% glycerol) with 10 mm MgCl2. To isolate the synaptic complex, 32P-labeled nicked RSS DNA (3000 cpm/μl) was incubated with RAG1 (16 μg/ml), RAG2 (16 μg/ml), and HMG1 (8 μg/ml) proteins at 37 °C for 10 min in the cleavage buffer containing 0.3 μm competitor DNA and 10 mm CaCl2. The biotinylated partner RSS (8 nm) was then added and further incubated at 37 °C for 90 min. To isolate the complex, streptavidin-coated magnetic beads (Dynabeads M-280; 10 μg/μl) in binding buffer were added, and the reaction mixture was incubated at 37 °C for 20 min. After incubation, magnetic beads and supernatant were separated using a magnet stand. The beads were washed three times at room temperature with 15 μl of binding buffer containing 10 mm CaCl2 and then resuspended in 30 μl of binding buffer containing 10 mm CaCl2. Footprinting–For DMS footprinting, 1.5 μl of 10% DMS (in ethanol) was added to the isolated complex (preheated at 25 °C for 2 min) and incubated at 25 °C for 6 min. Methylation was stopped by adding β-mercaptoethanol (0.2 m) and sodium acetate (0.3 mm). DNA was extracted with phenol/chloroform/isoamylalchohol (25:24:1), precipitated twice with ethanol, dissolved in 10 mm sodium phosphate (pH 6.8) and 1 mm EDTA, and heated at 90 °C for 15 min. Sodium hydroxide (0.1 n) was then added, and heated at 90 °C for 30 min. DNA was precipitated with ethanol, washed with 70% ethanol, dissolved in formamide dye mix, and electrophoresed in a 10% denaturing polyacrylamide gel. For KMnO4 footprinting, 32P-labeled RSS DNA (400 cpm/μl) and partner RSS DNA (8 nm) were incubated with RAG1 (12 μg/ml), RAG2 (12 μg/ml), and HMG1 (8 μg/ml) proteins at 37 °C for 45 min in cleavage buffer containing either 10 mm MgCl2 or 10 mm CaCl2. Samples were preheated at 25 °C for 5 min. KMnO4 (10 mm) was then added and further incubated at 25 °C for 6 min. Oxidation was stopped by adding β-mercaptoethanol (1.2 m). SDS (0.556%) and proteinase K (0.556 mg/ml) were added to the sample and incubated at 50 °C for 60 min. DNA was precipitated twice with ethanol, dissolved in 10% piperidine, heated at 90 °C for 30 min, and freeze-dried three times. Samples were dissolved in formamide dye mix and electrophoresed in a 10% denaturing polyacrylamide gel. RAG1 Mutants for Synapsis-dependent RSS Cleavage–To analyze the synapsis-dependent RSS cleavage, we tried to isolate the RAG1 mutants defective in partner-dependent hairpin formation. Mutants were generated by swapping short stretches of the murine RAG1 sequence with the corresponding regions from sea urchin proteins deduced from the RAG1-like genes (22Kapitonov V.V. Jurka J. PLoS Biol. 2005; 3: e181Crossref PubMed Scopus (365) Google Scholar, 23Fugmann S.D. Messier C. Novack L.A. Cameron R.A. Rast J.P. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3728-3733Crossref PubMed Scopus (137) Google Scholar, 24Sodergren E. Weinstock G.M. Davidson E.H. Cameron R.A. Gibbs R.A. Angerer R.C. Angerer L.M. Arnone M.I. Burgess D.R. Burke R.D. Coffman J.A. Dean M. Elphick M.R. Ettensohn C.A. Foltz K.R. Hamdoun A. Hynes R.O. Klein W.H. Marzluff W. McClay D.R. Morris R.L. Mushegian A. Rast J.P. Smith L.C. Thorndyke M.C. Vacquier V.D. Wessel G.M. Wray G. Zhang L. Elsik C.G. Ermolaeva O. Hlavina W. Hofmann G. Kitts P. Landrum M.J. Mackey A.J. Maglott D. Panopoulou G. Poustka A.J. Pruitt K. Sapojnikov V. Song X. Souvorov A. Solovyev V. Wei Z. Whittaker C.A. Worley K. Durbin K.J. Shen Y. 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With the HA3 mutant, where residues 978–984 were replaced, more than 75% of total 12-RSS was converted to the hairpin form, and the addition of the partner 23-RSS did not enhance the reaction (Fig. 1B). The HA3 phenotype is similar to that for the previously reported RAG1 mutant (E649A) that cleaves RSS DNA independent of the synapse formation (46Kriatchko A.N. Anderson D.K. Swanson P.C. Mol. Cell Biol. 2006; 26: 4712-4728Crossref PubMed Scopus (14) Google Scholar). Two other mutants, HA1 and HA2, demonstrated elevated levels of hairpin formation by severalfold in the absence of the partner RSS, which could be further enhanced with the addition of partner RSS (Fig. 1B). Another group of RAG1 mutants identified in the present study demonstrated defective hairpin formation (DH) even in the presence of partner RSS. With one of the mutants, DH3u, hairpin formation was not enhanced by the addition of the partner RSS, although the partner-independent basal hairpin formation activity was comparable with wild type (Fig. 1B). Under the relaxed condition using Mn2+ (27McBlane J.F. van Gent D.C. Ramsden D.A. Romeo C. Cuomo C.A. Gellert M. Oettinger M.A. Cell. 1995; 83: 387-395Abstract Full Text PDF PubMed Scopus (592) Google Scholar, 31Eastman Q.M. Leu T.M. Schatz D.G. Nature. 1996; 380: 85-88Crossref PubMed Scopus (211) Google Scholar, 47van Gent D.C. McBlane J.F. Ramsden D.A. Sadofsky M.J. Hesse J.E. Gellert M. Cell. 1995; 81: 925-934Abstract Full Text PDF PubMed Scopus (268) Google Scholar), instead of Mg2+, the DH3u cleaved RSS DNA as efficiently as the wild-type RAG1 (Fig. S1). RAG-7-Mer Interaction Is Enhanced by the Synapsis Formation–We then studied the RAG-RSS interaction at different stages of synaptic complex formation. Using the nicked RSS as an intermediary substrate, both the single RSS complex and the synaptic complex were analyzed in parallel by DMS footprinting (Figs. 2 and S2). Hairpin formation, which usually hampers the footprinting, was blocked by two different methods. First we utilized a catalytic mutant of RAG1 (D600A) that can form the RAG-RSS complex but not the hairpin structure (Fig. 2) (48Kim D.R. Dai Y. Mundy C.L. Yang W. Oettinger M.A. Genes Dev. 1999; 13: 3070-3080Crossref PubMed Scopus (177) Google Scholar, 49Fugmann S.D. Villey I.J. Ptaszek L.M. Schatz D.G. Mol. Cell. 2000; 5: 97-107Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In the other method, the 3′-OH at the nick was reduced to the 3′-dideoxy form, and the reaction was performed under the Ca2+ condition (Fig. S2) (43Nagawa F. Hirose S. Nishizumi H. Nishihara T. Sakano H. J. Biol. Chem. 2004; 279: 38360-38368Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In each experiment, either the 12- or 23-RSS was labeled with 32Patthe 5′-end of the bottom strand and incubated with HMG1 and GST-fused RAG1/2 proteins with or without the partner RSS. The resulting complex was isolated with glutathione-Sepharose (Figs. 2A and S2). The addition of the partner RSS satisfying the 12/23 rule increased the yield of the complex, indicat
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