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J. Biol. Chem., Vol. 276, Issue 37, 35024-35028, September 14, 2001

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A Unique -Hairpin Protruding from AAA⁺ ATPase Domain of RuvB Motor Protein Is Involved in the Interaction with RuvA DNA Recognition Protein for Branch Migration of Holliday Junctions^*

Yong-Woon Han, Hiroshi Iwasaki^§¶, Tomoko Miyata^**, Kouta Mayanagi^**, Kazuhiro Yamada^**, Kosuke Morikawa^**, and Hideo Shinagawa

From the  Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka, Suita, Osaka 565-0871, the ^§ Japan Science and Technology Corporation Precursory Research for Embryonic Science and Technology, 3-1 Yamadaoka, Suita, Osaka 565-0871, and the ^** Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan

Received for publication, April 23, 2001, and in revised form, June 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Escherichia coli RuvB protein is a motor protein that forms a complex with RuvA and promotes branch migration of Holliday junctions during homologous recombination. This study describes the characteristics of two RuvB mutants, I148T and I150T, that do not promote branch migration in the presence of RuvA. These RuvB mutants hydrolyzed ATP and bound duplex DNA with the same efficiency as wild-type RuvB, but the mutants did not form a complex with RuvA and were defective in loading onto junction DNA in a RuvA-assisted manner. A recent crystallographic study revealed that Ile¹⁴⁸ and Ile¹⁵⁰ are in a unique -hairpin that protrudes from the AAA⁺ ATPase domain of RuvB. We propose that this -hairpin interacts with hydrophobic residues in the mobile third domain of RuvA and that this interaction is vital for the RuvA-assisted loading of RuvB onto Holliday junction DNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Homologous recombination plays important biological roles in regulating genetic diversity and in repairing damaged chromosomes. Homologous recombination involves a series of enzymatic reactions carried out by large multiprotein complexes. One intermediate of homologous recombination is a four-stranded DNA structure called a Holliday junction (1). In bacteria, Holliday junctions are processed at a late stage of recombination into two recombinant duplex DNA molecules by a protein complex that includes RuvA, RuvB, and RuvC (2, 3).

RuvA and RuvB form a complex that promotes movement of a Holliday junction, a process known as branch migration. Electron microscopic studies have demonstrated that RuvB forms a hexameric ring that encircles duplex DNA (4). Two RuvB hexameric rings flank two RuvA tetramers that sandwich the Holliday junction (5). This structure suggests that homologous (homeologous) DNA duplexes are unwound and rewound during branch migration while they pass through the RuvB rings via RuvA tetramers; this process leads to the formation of heteroduplex DNA.

The RuvA tetramer is a junction-specific binding protein that interacts directly with RuvB and loads RuvB onto Holliday junctions. The RuvA monomer consists of three domains: domains I and II are involved in tetramer formation and Holliday junction recognition, respectively (6-8). Domain III is highly mobile, is connected with domain II via a flexible loop, and is involved in a specific interaction with RuvB (7, 9).

The RuvB hexamer is a motor that drives branch migration using energy derived from ATP hydrolysis (10, 11). The RuvB ATPase is synergistically stimulated by RuvA and DNA in vitro (12). RuvB can be dimeric, hexameric, heptameric, or dodecameric depending on conditions and cofactors such as ATP, Mg²⁺, and DNA (13-15). It also interacts with RuvC Holliday junction resolvase (16).

The RuvB protein is a member of the AAA⁺ class of ATPases (17). The crystal structure of RuvB from Thermus thermophilus HB8 was recently determined (18). This protein has a crescent-like architecture consisting of three consecutive domains. The first two domains have a folding pattern that is well conserved in AAA/AAA⁺ ATPases and is involved in ATP binding and hydrolysis. However, sequence alignments of AAA⁺ class proteins show that the amino acid sequence from Leu¹³⁵ to Leu¹⁵² in Escherichia coli RuvB is not conserved in other AAA/AAA⁺ class proteins such as N-ethylmaleimide-sensitive factor D2. This implies that this unique region is involved in a specific function of RuvB (17). This region forms -hairpin 1, which protrudes from the first domain of RuvB (Fig. 1) (18).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   A, alignment of AAA+ ATPase domains of E. coli RuvB, E. coli DNA polymerase III ' subunit (delta'), and N-ethylmaleimide-sensitive factor D2 (NSF D2). The predicted secondary structures of E. coli RuvB are indicated. Numbers in parentheses refer to inserted residues that are not shown. The black-boxed residues indicate motifs for the AAA+ ATPase family. The two arrows indicate Ile148 and Ile150 residues altered to Thr. The lines over residues show the regions of secondary structure, -helixes and -sheets. B, locations of Ile148 and Ile150 of E. coli RuvB from the crystal structure of T. thermophilus RuvB (18). The monomer is viewed from the ATP binding site. Domains I, II, and III are colored blue, yellow, and green, respectively. Ile148 and Ile150 are green. -Hairpin 1 and Walker A/B motifs are red and magenta, respectively.

This report describes the properties of two mutant RuvB proteins with mutations in -hairpin 1, I148T and I150T, which were isolated in a previous study (17). The two mutants have a similar phenotype in vivo and are defective in their functional and physical interactions with RuvA protein in vitro. We propose that residues Ile¹⁴⁸ and Ile¹⁵⁰ in -hairpin 1 interact with hydrophobic residues in the mobile domain III of RuvA and that this interaction is essential for RuvAB-dependent branch migration.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains, Plasmids, and Growth Conditions-- E. coli strain HRS3401 (ruvB::Km^r) is a derivative of AB1157 (19), a ruv⁺ strain. HRS4000, which is a ruvABC::Km^r derivative of BL21(DE3) (20), was used to overproduce mutant RuvB proteins. pAF101 (21), a derivative of pET3a (20), was used to overexpress wild-type and mutant ruvB and for purification of wild-type RuvB. pYWH500 (22), a derivative of pSTV28 (Takara Shuzo Co., Ltd.), was used for moderate expression of the ruvB gene and mutants. pET11a (20) was used for regulated low level expression of RuvB and for purification of mutant RuvB proteins. Bacteria were cultured in Luria-Bertani medium containing the appropriate antibiotics (23).

UV Light Sensitivity Test-- Exponentially growing AB1157 or HRS3401 (ruvB) cells harboring ruvB expression plasmids were suspended in M9 buffer (~2 × 10⁸ cells/ml) and irradiated with various doses of UV. Cells were plated on Luria-Bertani plates containing ampicillin (50 µg/ml), and the surviving colonies were scored after incubation for 16 h at 37 °C in the dark (24).

Protein Purification-- The wild-type and mutant RuvB proteins were overproduced in E. coli HRS4000 using a T7 expression system and purified essentially as described previously (24) except that hydrophobic interaction chromatography using RESOURCE PHE (Amersham Pharmacia Biotech) was added between ammonium sulfate precipitation and the first anion exchange column chromatography. Protein concentrations were determined using ₂₈₀ = 16,900 M¹ cm¹ (24).

Branch Migration Assay-- Branch migration assays were carried out as described previously (24) except that the reaction mixture (20 µl) contained 5 nM ³²P-labeled synthetic Holliday junction DNA, 20 mM Tris acetate (pH 8.0), 10 mM Mg(OAc)₂, 2 mM ATP, 1 mM dithiothreitol, and bovine serum albumin at 100 µg/ml.

ATPase Assays-- ATPase assays were carried out as described previously (24) except that the reaction mixtures contained 20 mM Tris acetate (pH 8.0), 10 mM Mg(OAc)₂, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, the indicated concentration of ATP, 100 µM form I pUC19 DNA, 0.6 µM RuvA protein, and 1 µM RuvB protein. The k_cat for ATP hydrolysis was calculated from double-reciprocal plots of initial rates of ATP hydrolysis as a function of increasing ATP concentration.

Assay for the Formation of RuvA, RuvB, and Holliday Junction Tripartite Complex-- Binding reactions (20 µl) contained 10 nM ³²P-labeled Holliday junction DNA, 20 mM triethanolamine-HCl (pH 7.5), 10 mM Mg(OAc)₂, 0.25 mM ATPS,¹ 1 mM dithiothreitol, and 50 µg/ml bovine serum albumin. The reactions were incubated for 20 min at 37 °C. Protein-DNA complexes were fixed by incubation with glutaraldehyde at 0.2% (v/v) at 37 °C for 30 min. Reaction products were analyzed by electrophoresis at 12.5 V/cm in a 6% polyacrylamide gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA) at room temperature.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RuvB I148T and I150T Partially Complement the DNA Repair Deficiency of ruvB Strain-- RuvB proteins I148T and I150T strongly inhibit cell growth when overexpressed from a high copy number plasmid (17). These constructs produce RuvB at a level ~200-fold higher than that produced from the chromosomal ruvB gene. However, if RuvB mutant proteins are expressed from a regulated promoter (pET11a) at a low level (3-fold higher than the level produced from the chromosomal ruvB gene), cell growth is not inhibited. Wild-type cells expressing RuvB I148T and I150T at this level formed normal colonies and had normal sensitivity to UV (data not shown); however, the UV sensitivity of a ruvB strain was not complemented by these proteins (Fig. 2A). RuvB I148T and I150T were expressed at a slightly higher level from the pSTV28 vector (~10-fold higher than the level produced from the chromosomal ruvB gene); at this level, RuvB I148T and I150T partially complemented the UV sensitivity of a ruvB deletion strain (Fig. 2B). These results suggest that RuvB I148T and I150T are less active in DNA repair than wild-type RuvB, but they have partial activity that is evident if they are moderately overexpressed.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   UV sensitivity of ruvB strain expressing the mutant genes at a low (A) or moderate (B) level. , vector; , ruvB+ plasmid; , I148T plasmid; , I150T plasmid.

Branch Migration Activity of RuvB I148T and I150T-- RuvB I148T and I150T were overexpressed and purified to more than 97% homogeneity (data not shown), and their branch migration activity was assessed. In the presence of ATP and RuvA, wild-type RuvB dissociated the Holliday junction efficiently, RuvB I148T was ~10-fold less active than wild-type RuvB, and RuvB I150T activity was barely detectable in this assay at the highest concentration examined (Fig. 3). Therefore, I148T and I150T mutant RuvB proteins are functionally defective in branch migration in vivo and in vitro.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Dissociation of Holliday junctions by mutant RuvB. Samples containing 5 nM Holliday junction DNA, 50 nM RuvA, and various concentrations of RuvB were incubated for 30 min at 37 °C, and the reaction products were analyzed by 9% polyacrylamide gel electrophoresis. Lane a, no RuvAB; lanes b, g, and l, 50 nM RuvB; lanes c, h, and m, 100 nM RuvB; lanes d, i, and n, 200 nM RuvB; lanes e, j, and o, 400 nM RuvB; lanes f, k, and p, 800 nM RuvB. WT, wild type.

ATPase Activity of I148T and I150T Is Not Stimulated by RuvA-- The ATPase activity of RuvB (Table I) is absolutely required for branch migration activity and is stimulated synergistically by DNA and RuvA (10, 11, 13). In the absence of RuvA, the ATPase activities of I148T and I150T were comparable with the intrinsic activity of wild-type RuvB whether in the absence (wild-type RuvB, K_cat = 1.6) or presence of DNA (wild-type RuvB, K_cat = 10.8). The ATPase activity of wild-type RuvB was ~10-fold higher in the presence of RuvA and double-stranded DNA (K_cat = 103.5) than in the presence of DNA alone and 2.6-fold higher in the presence of RuvA (K_cat = 4.2) than in its absence (K_cat = 1.6). In contrast, the ATPase of RuvB I148T and I150T increased only 1.8- and 1.4-fold, respectively, in the presence of RuvA and DNA, and the mutant activity did not increase in the presence of RuvA alone. Therefore, the stimulation of the ATPase activity of RuvB I148T and I150T by RuvA was greatly reduced, which suggests that the mutants have an impaired ability to interact with RuvA.

RuvB I148T and I150T Are Defective in Forming a Complex with RuvA-- The interactions between wild-type and mutant RuvB and RuvA were analyzed using a gel filtration assay. As shown in Fig. 4A, wild-type RuvA eluted as a tetramer at 120 kDa, and wild-type RuvB eluted as a dimer at 120 kDa. These data indicate that the Stokes radii of these proteins, especially RuvB, are larger than the radius expected for a spherical protein with the same molecular mass (13). Wild-type RuvA and wild-type RuvB formed a complex that eluted at 250 kDa, suggesting that a RuvA-RuvB hetero-oligomeric complex is formed as reported previously (13, 24). RuvB I148T and I150T eluted at 120 kDa (Fig. 4, B and C). However, when RuvA and RuvB I150T were allowed to interact, the proteins eluted at 120 kDa, which is the same position as the RuvA tetramer and the RuvB I150T dimer (Fig. 4C). When RuvB I148T was allowed to interact with RuvA, a portion of the protein eluted at the position of a RuvA-RuvB (I148T) hetero-oligomer, but most of the protein eluted at the position of RuvA tetramer and RuvB I148T dimer (Fig. 4B). These results suggest that RuvB I148T and I150T are defective in their interaction with RuvA in solution and that RuvB I150T is more deficient than RuvB I148T.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Gel filtration of wild-type and mutant RuvB. Gel filtration was carried out using Superdex 200 as described previously (24). Elution profiles of RuvA, RuvB, and RuvA-RuvB complex (RuvAB) with wild-type RuvB (A); RuvB I148T (B), and RuvB I150T (C).

The RuvA-RuvB Complex on a Holliday Junction DNA Substrate-- The above studies demonstrate that RuvB I148T and I150T are defective in interacting with RuvA in solution; however, it seemed possible that the RuvB mutants could form a complex with RuvA in the presence of a Holliday junction DNA. To test this idea, formation of the ternary complex (RuvA-RuvB-DNA), where the DNA was a synthetic Holliday junction, was assayed by gel electrophoresis. The mutant RuvB proteins formed a ternary complex less efficiently than wild-type RuvB; relative to wild-type RuvB, a 3-fold higher concentration of RuvB I148T and a 5-fold higher concentration of RuvB I150T were required to form an equivalent amount of ternary complex (Fig. 5). Therefore, RuvA loads RuvB I148T and I150T onto a Holliday junction less efficiently than it loads wild-type RuvB.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Formation of a RuvA-RuvB-Holliday junction ternary complex. Samples containing 50 nM RuvA and various concentrations of RuvB were incubated with 10 nM Holliday junction (HJ) DNA for 20 min at 37 °C. The ternary complex was cross-linked with glutaraldehyde for 30 min at 37 °C and analyzed by 6% polyacrylamide gel electrophoresis. Lane a, no protein; lane b, no RuvB; lanes c, i, and o, 25 nM RuvB; lanes d, j, and p, 37.5 nM RuvB; lanes e, k, and q, 50 nM RuvB; lanes f, l, and r, 100 nM RuvB; lanes g, m, and s,150 nM RuvB; lanes h, n, and t, 300 nM RuvB. WT, wild type.

The reduced ability of these mutant RuvB proteins to form the ternary complex may reflect lower RuvB-associated DNA binding activity. Therefore, the DNA binding activities of RuvB I148T and I150T were quantified using a gel retardation assay (Fig. 6). The mobility shift increased with increasing RuvB concentration, and no difference was observed in the DNA binding properties of wild-type and mutant RuvB. This result suggests that RuvB I148T and I150T have normal DNA binding activity, which is consistent with the observation that DNA stimulates the ATPase activity of the mutant proteins (Table I).

View larger version (50K): [in this window] [in a new window]   Fig. 6.   DNA binding activity of mutant RuvB proteins. Samples containing various concentrations of RuvB I148T and I150T were incubated with form I pUC19 DNA (30 µM) for 20 min at 37 °C, and the DNA-protein complex was analyzed by agarose gel electrophoresis after cross-linking with glutaraldehyde for 30 min at 37 °C as described previously (24). Lane a, no protein; lanes b, g, and l, 1 µM RuvB; lanes c, h, and m, 2 µM RuvB; lanes d, i, and n, 3 µM RuvB; lanes e, j, and o, 4 µM RuvB; lanes f, k, and p, 5 µM RuvB. WT, wild type.

It also seemed possible that the altered properties of RuvB I148T and I150T reflect a reduced ability to form a hexamer ring to encircle duplex DNA. However, this possibility is not consistent with the results of electron microscopy of the mutant proteins, which showed that RuvB I148T and I150T form a hexameric ring structure around DNA in the presence of Mg²⁺ and ATPS (Fig. 7).

View larger version (84K): [in this window] [in a new window]   Fig. 7.   Electron microscopic observation of wild-type RuvB (A), I148T (B), and I150T (C). RuvB-DNA complex was prepared as described previously (15). For negative staining, the sample was applied to a copper grid supporting a continuous thin carbon film and stained with three drops of 2% uranyl acetate. Images were recorded at 48,000 magnification on Fuji FG film using a Joel 100cx microscope. Scale bar indicates 500 Å. WT, wild type.

Eggleston et al. (16) have shown previously that RuvB interacts with RuvC. The ability of RuvB I148T and I150T to interact with RuvC was tested using a gel supershift assay. The results showed that the RuvB mutants interact normally with RuvC (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study examined the properties of RuvB I148T and I150T, which have mutations in -hairpin 1. DNA stimulated the ATPase activity of the mutant and wild-type RuvB proteins similarly, but RuvA (or RuvA and DNA) stimulated the ATPase activity of the mutants to a much lower extent than it stimulated that of wild-type RuvB (Table I). The RuvB mutants were also deficient in the ability to form a complex with RuvA or a ternary complex with RuvA and a Holliday junction (Figs. 4 and 5). RuvB I148T and I150T bound duplex DNA and formed hexameric rings (Figs. 6 and 7) and interacted with RuvC (data not shown) in a manner similar to wild-type RuvB. Thus, the interaction of RuvB I148T and I150T with RuvA, which is required for the elevated ATPase and branch migration activities of the RuvA-RuvB complex, is defective.

The data also indicate that RuvB I148T is less severely impaired than RuvB I150T in ternary complex formation, ATP hydrolysis, and branch migration. Complementation analysis also showed that RuvB I148T retained more UV repair activity than RuvB I150T when the proteins were expressed highly in the mutant cells (Fig. 2). These findings further support the idea that the mutants are defective in the interaction with RuvA, and the result of the complementation analysis can be explained by proposing that the decrease in affinity of the mutant proteins can be compensated for by an increase in their concentration.

The crystal structure of RuvB from T. thermophilus HB8 was recently determined (18). The RuvB monomer consists of three domains (I, II, and III) that form a crescent-shaped configuration (Fig. 1B). The RuvB-specific region (L¹³⁵-L¹⁵²) forms -hairpin 1, which is composed of the fourth and fifth -strands. This -hairpin protrudes from the AAA⁺ ATPase motif in domain I (18). Leu¹⁴⁸ and Leu¹⁵⁰ are located in the fifth -strand (5). Electron microscopic studies demonstrated that the RuvB hexameric ring includes a large tier and a small tier, and the large tier faces RuvA (4, 5, 15). A tentative model of the hexameric ring based on the crystal structure shows that all six -hairpin 1 motifs are located on the top of the large tier (18). This is consistent with the idea that -hairpin 1 is involved in the interface between RuvB and RuvA.

It is particularly intriguing that ruvA mutations in hydrophobic residues such as Leu¹⁶⁷, Leu¹⁷⁰, Tyr¹⁷², and Leu¹⁹⁹ cause a defect in the RuvA-RuvB interaction. These residues are in mobile domain III of RuvA, which interacts specifically with RuvB (7, 9). Hydrophobic residues are well conserved in these positions involved in this interaction (7, 17). Therefore, the protruding -hairpin 1 in the AAA⁺ ATPase domain of RuvB may interact with hydrophobic residues in domain III of RuvA.

The mobile domain III of RuvA has also been shown not only to interact physically with RuvB but also to modulate RuvB ATPase activity. This suggests that the signal by RuvA for interaction with DNA may be transduced through the NH₂ region (domains I + II) to domain III of RuvA, resulting in continuous cycling of RuvB ATP hydrolysis (7, 9). Likewise, such a signal may also be transduced through domain III of RuvA to -hairpin 1 in domain I of RuvB. -Hairpin 1 of RuvB is situated between the fourth -helix and the sixth -sheet (18). It has been proposed that the fourth -helix is involved in intersubunit interaction, which may couple ATP binding or hydrolysis, and that the sixth -sheet is involved in sensing the ATP hydrolysis status of its own subunit (17, 18). Therefore, not only is -hairpin 1 involved in the physical interaction per se with RuvA, but also the interaction with RuvA via -hairpin 1 may cause a structural change of the ATPase domain of RuvB leading to efficient ATPase cycling. These physical and functional interactions may result in the regulation of RuvB motor activity to drive branch migration of the Holliday junction processively. We propose that -hairpin 1 interacts with RuvA in a structural and regulatory manner: this interaction may change the structure and activity of the RuvB ATPase domain and drive processive branch migration of Holliday junctions.

	FOOTNOTES

^* This work was supported by Grants-in-aid for Scientific Research 08280102 and 0828010 on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan (to H. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed: Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Tel.: 81-45-508-7238; Fax: 81-45-508-7369; E-mail: iwasaki@tsurumi.yokohama-cu.ac.jp.

To whom correspondence may be addressed. Tel.: 81-6-6879-8317; Fax: 81-6-6879-8320; E-mail: shinagaw@biken.osaka-u.ac.jp.

Published, JBC Papers in Press, June 26, 2001, DOI 10.1074/jbc.M103611200

	ABBREVIATIONS

The abbreviation used is: ATPS, adenosine 5'-O-(thiotriphosphate).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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