Structure-Function Analysis of the Three Domains of RuvB DNA Motor Protein
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m502400200
ISSN1083-351X
AutoresTakayuki Ohnishi, Takashi Hishida, Yoshie Harada, Hiroshi Iwasaki, Hideo Shinagawa,
Tópico(s)RNA Interference and Gene Delivery
ResumoRuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex. RuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex. Homologous recombination enhances genetic diversity, contributes to genomic stability, and plays an important role in DNA repair. In prokaryotes, RuvA, RuvB, and RuvC proteins process a central intermediate of homologous recombination, the Holliday junction, in a late stage of homologous recombination (1Holliday R. Genet. Res. 1964; 5: 282-304Crossref Scopus (1260) Google Scholar, 2Shinagawa H. Iwasaki H. Trends Biochem. Sci. 1996; 21: 107-111Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 3West S.C. Annu. Rev. Genet. 1997; 31: 213-244Crossref PubMed Scopus (386) Google Scholar). RuvA and RuvB form a complex that promotes branch migration of the Holliday junction. The RuvA tetramer, a Holliday junction-specific binding protein, facilitates the binding of RuvB to the Holliday junction. Two RuvA tetramers bind to opposite sides of the Holliday junction, and RuvB hexamers are tethered to and flank the RuvA tetramers (4Stasiak A. Tsaneva I.R. West S.C. Benson C.J. Yu X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Crossref PubMed Scopus (143) Google Scholar, 5Roe S.M. Barlow T. Brown T. Oram M. Keeley A. Tsaneva I.R. Pearl L.H. Mol. Cell. 1998; 2: 361-372Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6Privezentzev C.V. Keeley A. Sigala B. Tsaneva I.R. J. Biol. Chem. 2005; 280: 3365-3375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The RuvB hexamer forms a ring that encircles double-stranded DNA (dsDNA) 1The abbreviations used are: dsDNA, double-stranded DNA; Tth, Thermus thermophilus; AAA, ATPase associated with various cellular activities; GST, glutathione S-transferase; HJ, Holliday junction; ATPγS, adenosine-5′-O-(3-thiotriphosphate). 1The abbreviations used are: dsDNA, double-stranded DNA; Tth, Thermus thermophilus; AAA, ATPase associated with various cellular activities; GST, glutathione S-transferase; HJ, Holliday junction; ATPγS, adenosine-5′-O-(3-thiotriphosphate). and promotes DNA translocation in an ATP hydrolysis-dependent manner (2Shinagawa H. Iwasaki H. Trends Biochem. Sci. 1996; 21: 107-111Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 3West S.C. Annu. Rev. Genet. 1997; 31: 213-244Crossref PubMed Scopus (386) Google Scholar). RuvC is a structure-specific endonuclease that resolves the Holliday junction (7Dunderdale H.J. Benson F.E. Parsons C.A. Sharples G.J. Lloyd R.G. West S.C. Nature. 1991; 354: 506-510Crossref PubMed Scopus (193) Google Scholar, 8Iwasaki H. Takahagi M. Shiba T. Nakata A. Shinagawa H. EMBO J. 1991; 10: 4381-4389Crossref PubMed Scopus (222) Google Scholar). The nicked ends of the two DNA molecules are sealed by DNA ligase to complete the recombination reaction.RuvB has intrinsic ATPase activity (9Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2200-2214Crossref Scopus (148) Google Scholar, 10Tsaneva I.R. Müller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar) that is synergistically enhanced by RuvA and DNA (11Shiba T. Iwasaki H. Nakata A. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8445-8449Crossref PubMed Scopus (94) Google Scholar). RuvB binds to DNA in the presence of ATPγS in vitro (12Müller B. Tsaneva I.R. West S.C. J. Biol. Chem. 1993; 268: 17185-17189Abstract Full Text PDF PubMed Google Scholar). The structural analysis of RuvB suggests that it is a member of the AAA+ (ATPase associated with various cellular activities) ATPase superfamily rather than a member of the hexameric helicase family as previously suggested (13Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar, 14Iwasaki H. Han Y.W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). The crystal structures of RuvB from Thermus thermophilus HB8 (Tth) and Thermotoga maritima were recently solved (15Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 16Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (145) Google Scholar). These structures and other data suggest that the structure and function of the RuvAB complex are highly conserved among bacteria. The RuvB monomer has distinct amino-terminal (N), central (M), and carboxyl-terminal (C) domains (see Fig. 1A). The N and M domains are structurally similar to equivalent domains in other AAA+ family ATPases, except that a unique β-hairpin protrudes from the RuvB domain N. This β-hairpin physically interacts with RuvA and is required for formation of the RuvAB complex (17Nishino T. Iwasaki H. Kataoka M. Ariyoshi M. Fujita T. Shinagawa H. Morikawa K. J. Mol. Biol. 2000; 298: 407-416Crossref PubMed Scopus (27) Google Scholar, 18Han Y.W. Iwasaki H. Miyata T. Mayanagi K. Yamada K. Morikawa K. Shinagawa H. J. Biol. Chem. 2001; 276: 35024-35028Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19Yamada K. Miyata T. Tsuchiya D. Oyama T. Fujiwara Y. Ohnishi T. Iwasaki H. Shinagawa H. Ariyoshi M. Mayanagi K. Morikawa K. Mol. Cell. 2002; 10: 671-681Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Domain C of RuvB contains a winged helix motif, which is topologically similar to the DNA-binding domains of metallothionein repressor SmtB and histone H5 (15Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 16Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (145) Google Scholar). Structural and mutational analyses of RuvB suggest that it contains several functional motifs related to ATPase activity, including Walker motifs, sensor motifs, and an arginine finger (14Iwasaki H. Han Y.W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar, 15Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 16Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (145) Google Scholar, 20Hishida T. Han Y.W. Fujimoto S. Iwasaki H. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9573-9577Crossref PubMed Scopus (35) Google Scholar). RuvB is a multifunctional protein that interacts with RuvA, ATP, DNA, and Mg2+. These interactions and RuvB ATPase are required for RuvAB-dependent branch migration of Holliday junctions.This study analyzes the function of seven RuvB mutants, five truncated RuvB mutants lacking one or more RuvB domains and two RuvB point mutants with amino acid substitution mutations of Arg-318, a C-domain residue that plays a role in DNA binding (15Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). The results provide insight into the specific functional roles of each of the three RuvB domains. Furthermore, the data presented here suggest that coordinated ATP-dependent functions of the N, M, and C domains of RuvB are required for DNA binding, ATP hydrolysis, and RuvAB-catalyzed branch migration of Holliday junctions.EXPERIMENTAL PROCEDURESEscherichia coli Strains and Plasmids—HRS2301 (ΔruvB::Cmr) (21Hishida T. Iwasaki H. Ishioka K. Shinagawa H. Gene (Amst.). 1996; 182: 63-70Crossref PubMed Scopus (20) Google Scholar), derived from AB1157 (22Bachmann B.J. Bacteriol. Rev. 1972; 36: 525-557Crossref PubMed Google Scholar), was used as a ΔruvB strain for the UV light sensitivity test. BL21 (DE3) (23Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar) was used for overexpression of the GST fusion proteins. HRS4000 (ΔruvABC::Kmr) (24Yamada K. Fukuoh A. Iwasaki H. Shinagawa H. Mol. Gen. Genet. 1999; 261: 1001-1011Crossref PubMed Scopus (15) Google Scholar), derived from BL21 (DE3), was used for overexpression of the recombinant RuvA and RuvB proteins. pAF101 was used for overexpression of wild-type and mutant ruvB genes (24Yamada K. Fukuoh A. Iwasaki H. Shinagawa H. Mol. Gen. Genet. 1999; 261: 1001-1011Crossref PubMed Scopus (15) Google Scholar). The plasmid pRB100 carrying the wild-type ruvB gene is a derivative of pAF101 (24Yamada K. Fukuoh A. Iwasaki H. Shinagawa H. Mol. Gen. Genet. 1999; 261: 1001-1011Crossref PubMed Scopus (15) Google Scholar). pRB701, pRB702, pRB703, pRB704, and pRB705 encode ruvB-N, ruvB-NM, ruvB-M, ruvB-MC, and ruvB-C, respectively. pRB529 and pRB530 carry R318K and R318A mutant ruvB genes, respectively. pGE1 was derived from the GST fusion vector pGEX6p-1 (Amersham Biosciences) (19Yamada K. Miyata T. Tsuchiya D. Oyama T. Fujiwara Y. Ohnishi T. Iwasaki H. Shinagawa H. Ariyoshi M. Mayanagi K. Morikawa K. Mol. Cell. 2002; 10: 671-681Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). pGB500, pGB501, pGB502, pGB503, pGB504, and pGB505 encode proteins with GST fused to wild-type RuvB, RuvB-N, RuvB-NM, RuvB-M, RuvB-MC, and RuvB-C, respectively.Design of the Truncated ruvB Mutants—The truncated ruvB genes were generated by PCR using the two primers described below, and the amplified fragments were ligated into pRB100 or pGE1. The following six primers were used to generate the truncated ruvB mutants: f10, 5′-GACCACAACGGTTTCCCTCT-3′; r1, 5′-ATATTGCAGATCGGATCCTTAATAGAATTC-3′; r2, 5′-CAGCAAAAGCTTGGATCCTTAATAATCGAA-3′; f1, 5′-GTGCAACGTCTGGAACATATGCAGGTGCCGG-3′; f2, 5′-TTGAATGTCCATATGGAAGGTTTCGATTAT-3′; t10, 5′GCTAGTTATTGCTCAGCGG-3′. The pairs of DNA primers used to generate were as follows: f10 and r1 for ruvB-N, f10 and r2 for ruvB-NM, f1 and t2 for ruvB-M, f1 and t10 for ruvB-MC, and f2 and t10 for ruvB-C.Site-directed Mutagenesis—Site-directed mutagenesis of Arg-318 was carried out as described previously (14Iwasaki H. Han Y.W. Okamoto T. Ohnishi T. Yoshikawa M. Yamada K. Toh H. Daiyasu H. Ogura T. Shinagawa H. Mol. Microbiol. 2000; 36: 528-538Crossref PubMed Scopus (38) Google Scholar). A pair of synthetic oligonucleotide primers, 5′-ACACCGCGTGGGAAAATGGCGACGACGCGG-3′ and 5′-CCGCGTCGTCGCCATTTTCCCACGCGGTGT-3′ were used to generate the ruvB R318K mutant (altered sequences for mutagenesis are underlined). A pair of synthetic oligonucleotide primers, 5′-ACACCGCGTGGGGCTATGGCGACGACGCGG-3′ and 5′-CCGCGTCGTCGCCATAGCCCCACGCGGTGT-3′ were used to generate the ruvB R318A mutant. The mutations were confirmed by DNA sequencing.UV Light Sensitivity Assay—pRB100 derivatives expressing the truncated RuvB proteins were introduced into HRS2301 or wild-type strain. Transformants were grown to stationary phase, and 10-fold serial dilutions of the cultures were spotted onto LB agar plates containing ampicillin at 100 μg/ml. The agar plates were irradiated by UV light at 45 J/m2 and incubated overnight at 37 or 26 °C.Protein Purification—The RuvA, wild-type RuvB, and RuvB R318A proteins were prepared as described (18Han Y.W. Iwasaki H. Miyata T. Mayanagi K. Yamada K. Morikawa K. Shinagawa H. J. Biol. Chem. 2001; 276: 35024-35028Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25Ariyoshi M. Vassylyev D.G. Iwasaki H. Nakamura H. Shinagawa H. Morikawa K. Cell. 1994; 78: 1063-1072Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 26Nishino T. Ariyoshi M. Iwasaki H. Shinagawa H. Morikawa K. Structure. 1998; 6: 11-21Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Ohnishi T. Iwasaki H. Ishino Y. Kuramitsu S. Nakata A. Shinagawa H. Genes Genet. Syst. 2000; 75: 233-243Crossref PubMed Scopus (8) Google Scholar). HRS4000 was used as a host for overproduction of the GST fusion RuvB proteins. HRS4000 carrying wild type or the truncated ruvB plasmids were grown at 37 °C in 2 liters of LB medium containing 100 mg/ml ampicillin. When reached A600 = 0.3, isopropyl-1-thio-β-d-galactopyranoside was added to a concentration of 2 mm for induction of the GST fusion RuvB protein synthesis, and culture was grown for another 6 h at 26 °C. The cells were collected by centrifugation and resuspended in R buffer (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 10% glycerol, 2 mm 2-mercaptoethanol) containing 0.1 m NaCl. After sonication and centrifugation, the supernatant was applied to a glutathione-Sepharose column (Amersham Biosciences) and eluted with elution buffer (50 mm Tris-HCl, pH 8.0, 10 mm reduced glutathione). GST was removed from the fusion protein using precision protease (Amersham Biosciences). The samples were dialyzed against storage buffer (10 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 50% glycerol) and stored at -30 °C. Protein concentrations were determined using a Bio-Rad protein assay kit.Branch Migration and ATPase Assay—The branch migration assay was carried out as described previously (18Han Y.W. Iwasaki H. Miyata T. Mayanagi K. Yamada K. Morikawa K. Shinagawa H. J. Biol. Chem. 2001; 276: 35024-35028Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20Hishida T. Han Y.W. Fujimoto S. Iwasaki H. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9573-9577Crossref PubMed Scopus (35) Google Scholar, 29Hishida T. Iwasaki H. Han Y.W. Ohnishi T. Shinagawa H. Genes Cells. 2003; 8: 721-730Crossref PubMed Scopus (6) Google Scholar). The substrate used for the branch migration assay was a 32P-labeled synthetic Holliday junction DNA (27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The reaction mixture (20 μl) was incubated for 30 min at 26 or 37 °C, and the reaction was stopped by the addition of 5 μl of stop buffer. The products were analyzed by PAGE in a 9% gel and visualized by autoradiography. The ATPase assay was performed as described previously (27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).DNA Binding Assay—The DNA binding assay was performed essentially as described (30Hishida T. Han Y.W. Shibata T. Kubota Y. Ishino Y. Iwasaki H. Shinagawa H. Genes Dev. 2004; 18: 1886-1897Crossref PubMed Scopus (111) Google Scholar). The standard reaction mixture (20 μl) contained 20 mm Tris acetate (pH 8.0), 10 mm Mg(OAc)2, 0.01% bovine serum albumin, 1 mm ATPγS, 10 nm 70-mer duplex DNA labeled with 32P, and wild type or truncated RuvB at the indicated concentration. Samples were incubated at 26 or 37 °C for 30 min and analyzed by PAGE in a 6% gel in TAM buffer (40 mm Tris acetate (pH 7.8), 0.5 mm Mg(OAc)2), followed by visualization by autoradiography.Formation of RuvAB Holliday Junction Complex—The assay was carried out as described previously (18Han Y.W. Iwasaki H. Miyata T. Mayanagi K. Yamada K. Morikawa K. Shinagawa H. J. Biol. Chem. 2001; 276: 35024-35028Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 31Parsons C.A. West S.C. J. Mol. Biol. 1993; 232: 397-405Crossref PubMed Scopus (82) Google Scholar). The reaction mixture (20 μl) was incubated for 20 min at 26 °C. Protein-DNA complexes were fixed by incubation with 0.2% glutaraldehyde at 26 °C for 30 min. The reaction products were analyzed by PAGE in a 6% gel and visualized by autoradiography.Gel Filtration Analysis of RuvB Oligomer and RuvAB Complex Formation—Gel filtration experiments to assess RuvAB protein complex formation were carried out as described previously (27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Samples of RuvA (15 μm), RuvB (10 μm), and a mixture of RuvA and RuvB were applied to a Superdex-200 column equilibrated with buffer A (20 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 50 mm NaCl, and 5% glycerol). RuvB samples contained intact RuvB, RuvB-N, or RuvB-NM. The truncated RuvBs containing RuvB-M, RuvB-MC, and RuvB-C were applied to a Superdex-75 column. Protein peaks were monitored by absorbance at the wavelengths 225 and 280 nm. The assay to examine RuvB hexamer formation was carried out as described previously (27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The wild type and mutant RuvBs (35 μm) were incubated for 3 h at 4 °C in buffer A containing 10 mm MgCl2 and 0.25 mm ATP and 10-μl aliquots were applied to the column equilibrated with buffer A containing 10 mm MgCl2 and 0.25 mm ATP.Glycerol Gradient Sedimentation Assay—Glycerol gradient sedimentation was carried out as described previously (32Ichiyanagi K. Iwasaki H. Hishida T. Shinagawa H. Genes Cells. 1998; 3: 575-586Crossref PubMed Scopus (16) Google Scholar). The linear glycerol gradient from 15 to 35% in the same buffer as that used for gel filtration. Marker proteins for molecular size, Stokes' radii, and sedimentation coefficiency were as follows: aldolase (158 kDa, 48.1 Å, 7.6 S), bovine serum albumin (67 kDa, 35.5 Å, 4.31 S), ovalbumin (43 kDa, 30.5 Å, 3.66 S), chymotrypsinogen A (25 kDa, 20.9 Å, 2.55 S), ribonuclease A (13.7 kDa, 16.4 Å), and aprotinin (6.5 kDa). For calculation of native molecular sizes by the Siegel-Montry equation (33Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar), we employed an average partial specific volume of 0.73 cm3/g.RESULTSStructure-Function Analysis of RuvB Using Domain Truncations—RuvB has an N, M, and a C domain (15Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar) (Fig. 1A) and each of these domains may have distinct or overlapping functional and/or structural roles in RuvB. The role(s) of each RuvB domain was examined using ruvB mutants that lack one or more of these domains (Fig. 1B). These mutants are called RuvB-N, -NM, -M, -MC, and -C, where the letter indicates the intact domain(s) of the truncated variant. RuvB mutants were tested for complementation of the DNA repair deficiency of E. coli strain HRS2300 (ΔruvB) and for dominant negative character in wild-type AB1157 at 37 or 26 °C. Repair deficiency was assayed by treating wild type and mutant cells with or without UV irradiation. Truncated mutants were expressed in vivo from high copy number expression plasmids constructed with vector pAF101 (see "Experimental Procedures").Fig. 1, C and D, shows the characteristics of each RuvB truncation mutant. None of the RuvB truncation mutants complemented the UV light sensitivity phenotype of ΔruvB at 37 or 26 °C (Fig. 1C), but the positive control (wild-type RuvB) did complement the DNA repair defect. In addition, RuvB-N and RuvB-NM demonstrated dominant negative effects on wild-type cells, and the dominant negative phenotype was more severe at 26 than at 37 °C (Fig. 1D). The dominant negative phenotype was stronger in cells overexpressing RuvB-NM than in cells overexpressing RuvB-N. RuvB-M, RuvB-MC, and RuvB-C had no dominant negative effects on wild-type cells at 37 or 26 °C.Domain Requirements for RuvB ATPase—To examine the biochemical properties of the mutant RuvB proteins, truncated RuvB mutants were purified by affinity chromatography as GST fusion proteins. After affinity chromatography, the recombinant RuvB moiety was separated from the GST moiety using precision protease cleavage of the fusion protein linker. When overexpressed in E. coli at 37 °C, RuvB-NM and RuvB-N were largely insoluble. However, the solubility of these proteins was significantly higher when they were overexpressed at 26 instead of 37 °C (data not shown), suggesting that the mutant proteins may fold incorrectly at the higher temperature. This result is consistent with the observation that the dominant negative effects of RuvB-NM and RuvB-N are more severe at 26 than at 37 °C.RuvB ATPase activity is required for RuvAB-catalyzed branch migration at Holliday junctions. RuvB ATPase is stimulated by the addition of RuvA or DNA and simultaneous addition of RuvA and DNA synergistically activates the RuvB ATPase (9Iwasaki H. Takahagi M. Nakata A. Shinagawa H. Genes Dev. 1992; 6: 2200-2214Crossref Scopus (148) Google Scholar, 34Tsaneva I.R. Illing G. Lloyd R.G. West S.C. Mol. Gen. Genet. 1992; 235: 1-10Crossref PubMed Scopus (87) Google Scholar, 35Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1993; 237: 395-399Crossref PubMed Scopus (40) Google Scholar). Fig. 2A shows the effect of domain truncation on RuvB ATPase activity at 37 °C. Unlike wild-type RuvB, RuvB-NM ATPase is stimulated equally by RuvA or RuvA-DNA and is not stimulated by DNA alone. RuvB-NM also has a temperature-sensitive ATPase that is much weaker than the ATPase of wild-type RuvB at 37 °C, but equivalent to the basal wild-type ATPase at 26 °C (Fig. 2B). This result may reflect the higher solubility of RuvB-NM at 26 than at 37 °C. RuvB-N, -M, -MC, and -C had no detectable ATPase activity at 26 °C (Fig. 2C), suggesting that domains N and M are each required but neither is sufficient for RuvB ATPase.Fig. 2ATPase activity of wild-type RuvB and the truncated RuvB mutants. A, ATPase assays were carried out in the presence of 1 μm wild-type RuvB or RuvB-NM in the absence of DNA and RuvA (diamond), in the presence of 0.6 μm RuvA (square), in the presence of 100 μm (nucleotide moles) supercoiled DNA (triangle) or in the presence of RuvA and supercoiled DNA (circle) for the indicated times at 37 °C. B, reactions were carried out in the presence of 1 μm wild-type RuvB, or RuvB-NM in the absence of DNA and RuvA for 60 min at 37 or at 26 °C. Ratio of ATPase activity of RuvB-NM relative to those of wild-type RuvB are shown. The actual values of ATP hydrolysis determined for the wild-type controls at 37 and 26 °C were 1.8 and 0.8 nm, respectively. C, reactions were carried out in the presence of 1 μm wild-type or truncated RuvB mutants in the absence of DNA and RuvA at 26 °C. The data are means of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Domain Requirements for RuvB Branch Migration—The capacity of RuvB truncation mutants to carry out branch migration in vitro was assessed using a synthetic Holliday junction as a substrate. Wild-type RuvB dissociated the Holliday junction efficiently at 26 °C in the presence of ATP and RuvA; in contrast, none of the RuvB truncation mutants were capable of promoting branch migration or dissociating the synthetic Holliday junction under identical conditions (Fig. 3A). Furthermore, RuvB-N and RuvB-NM inhibited wild-type RuvAB-catalyzed branch migration (Fig. 3B, lanes h-t). This result may reflect the same mechanism that leads to the dominant negative phenotype of RuvB-N and RuvB-NM with respect to repair of UV-induced DNA damage in vivo.Fig. 3Branch migration activity of truncated RuvB mutants. A, RuvB was incubated at various concentration (50, 100, and 200 nm) with a synthetic Holliday junction (10 nm in Holliday junction moles) and 50 nm RuvA. Reactions were carried out at 26 °C for 30 min. The reaction products were analyzed by 9% PAGE and visualized by autoradiography. WT, wild type. B, inhibition of branch migration activity of wild-type RuvB by the truncated RuvB mutants. Branch migration activity was assayed by incubating 10 nm synthetic Holliday junctions (HJ) with 50 nm RuvA and the indicated concentration of RuvB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interaction between Truncated RuvB Mutants and RuvA—The capacity of RuvB truncation mutants to interact with RuvA was examined using gel filtration analysis in the presence or absence of ATP and Mg2+. In the absence of ATP and Mg2+, RuvA, wild-type RuvB and the RuvAB complex eluted at positions of 120, 100, and 280 kDa (Fig. 4A), respectively (27Hishida T. Iwasaki H. Yagi T. Shinagawa H. J. Biol. Chem. 1999; 274: 25335-25342Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). RuvB-N eluted at 70 kDa and the RuvB-N complex with RuvA eluted at 190 kDa (Fig. 4B). RuvB-NM eluted at 90 kDa and the RuvB-NM complex with RuvA eluted at 250 kDa (Fig. 4C). These results indicate that RuvB-N and RuvB-NM form complexes with RuvA. RuvB-M, RuvB-MC, and RuvB-C eluted at 6.7, 6.9, and 5.8 kDa, which may correspond to their predicted molecular sizes of 8, 17, and 9 kDa, suggesting that these proteins probably exist in solution as monomers. The elution of RuvB-MC at 6.9 kDa seems somewhat anomalous; however, the sedimentation coefficiency of RuvB-MC was estimated to be 2.24 S, which predicts a native molecular size of 12 kDa (data not shown). RuvB-MC may take a structure deviated from a sphere in solution. None of the three RuvB truncation mutants, RuvB-M, RuvB-MC, and RuvB-C, showed any shift in the elution positions by the addition of RuvA (Fig. 4, D-F), indicating that they do not form complexes with RuvA.Fig. 4Interaction of the truncated RuvB mutants with RuvA. RuvB was incubated with RuvA in the absence of ATP and Mg2+ and analyzed by gel filtration. Gel filtration chromatography of wild-type (WT) RuvB (A), RuvB-N (B), and RuvB-NM (C) was performed with Superdex-200. Blue dextran (void volume), ferritin (440 kDa), catalase (252 kDa) aldorase (158 kDa), bovine serum albumin (67 kDa), and chymotrypsinogen A (25 kDa) were eluted at 31.2, 38.6, 43.8, 47.0, 53.1, and 61.4 min, respectively. Gel filtration chromatography of RuvB-M (D), RuvB-MC (E), and RuvB-C (F) was performed with Superdex-75. Blue dextran (void volume), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa) were eluted at 30.5, 35.5, 43.4, 46.0, and 55.7 min, respectively. The gel filtration profiles (Superdex-200) of truncated RuvB mutants in the presence of 10 mm MgCl2 and 0.25 mm ATP are shown in (G).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Domain N Is Required for RuvB-RuvB Intersubunit Interaction—The results of gel filtration analysis of RuvB, RuvB-N, and RuvB-NM indicate that they form oligomers larger than a monomer. Previous sedimentation analysis indicated that RuvB exists as a dimer in the absence of ATP and Mg2+ (35Shiba T. Iwasaki H. Nakata A. Shinagawa H. Mol. Gen. Genet. 1993; 237: 395-399Crossref PubMed Scopus (40) Google Scholar). Glycerol gradient sedimentation analysis was used here to calculate the native molecular size of RuvB truncation variants by the method of Siegel and Monty (33Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar) (Table I). These data indicate that RuvB, RuvB-N, and RuvB-NM form dimers with the calculated native molecular sizes of 74, 39, and 59 kDa, respectively. These results suggest that domain N includes an interface required for RuvB dimer formation in the absence of ATP and Mg2+.Table IEstimation of the molecular mass of RuvB mutantsStokes' radiusaKav was
Referência(s)