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J. Biol. Chem., Vol. 276, Issue 38, 35735-35740, September 21, 2001

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Dissection of the Regional Roles of the Archaeal Holliday Junction Resolvase Hjc by Structural and Mutational Analyses^*

Tatsuya Nishino, Kayoko Komori^§, Yoshizumi Ishino^§, and Kosuke Morikawa^¶

From the Department of Structural Biology and ^§ Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan

Received for publication, May 16, 2001, and in revised form, June 28, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hjc is an archaeal DNA endonuclease, which resolves the Holliday junction in the presence of divalent metals. Combined with mutational analyses, the x-ray structure of the Pyrococcus furiosus Hjc crystal grown in the presence of ammonium sulfate revealed a positively charged interface, rich in conserved basic residues, and the catalytic center (Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K. (2001) Structure 9, 197-T204). This structural study also suggested that the N-terminal segment and some loops of Hjc play crucial roles in the cleavage of DNA. However, a structural view of the interaction between these regions and DNA remains elusive. To clarify the regional roles of Hjc in the recognition of the Holliday junction, further structural and biochemical analyses were carried out. A new crystal form of Hjc was obtained from a polyethylene glycol solution in the absence of ammonium sulfate, and its structure has been determined at 2.16-Å resolution. A comparison of the two crystal structures has revealed that the N-terminal segment undergoes a serious conformational change. The site-directed mutagenesis of the sulfate-binding site within the segment caused a dramatic decrease in the junction binding, but the mutant was still capable of cleaving DNA with a 20-fold lower efficiency. The kinetic analysis of Hjc-Holliday junction interaction indicated that mutations in the N-terminal segment greatly increased the dissociation rate constants of the Hjc-Holliday junction complex, explaining the decreased stability of the complex. This segment is also responsible for the disruption of base pairs near the junction center, through specific interactions with them. Taken together, these results imply that, in addition to the secondary effects of two basic loops, the flexible N-terminal segment plays predominant roles in the recognition of DNA conformation near the crossover and in correct positioning of the cleavage site to the catalytic center of the Hjc resolvase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Holliday junction is a universal DNA intermediate in homologous recombination, which plays crucial roles in the determination of genetic diversity and the repair of damaged chromosomes (1). This four-way junction DNA is formed during the exchange between two homologous DNA duplexes. The recombination process is terminated by symmetrical resolution of the junction, mediated by Holliday structure-specific endonucleases, which are found ubiquitously from bacteriophages to yeasts (reviewed in Refs. 2-4). The same activity is also present in mammalian cells, although the corresponding enzymes have not been identified (5, 6). These Holliday junction resolvases possess similar properties; they function as dimers and introduce symmetrical nicks into the Holliday junction in a divalent metal-dependent manner. They contain three or four invariant acidic residues that are important for the catalytic activity, in addition to several basic residues. Although these resolvases exhibit almost no sequence similarity, recent sequence and three-dimensional structural analyses have revealed that they can be classified into several distinct evolutionary families (7, 8). RuvC, Cce1, and pox A22 belong to the RNase H family containing a five-stranded -sheet structure flanked by -helices (9, 10). T7 endonuclease I and Hjc belong to the nuclease family, whose folding motif is similar to that of the type II restriction endonucleases (11, 12). T4 endonuclease VII contains a zinc-binding domain found in the McrA-like restriction endonuclease (13). The three-dimensional structure of RusA has been neither identified nor predicted. RuvC, CceI, and RusA exhibit a sequence preference upon cleavage, whereas T4 endonuclease VII, T7 endonuclease I, and Hjc appear to recognize junction structures (reviewed in Ref. 4). The Holliday junction assumes different conformations in the presence and absence of metal ions. Comparative gel electrophoresis revealed that the Holliday junction by itself adopts two different conformations: open square and stacked-X (14). These conformations are known to be rearranged upon binding to the resolvase (2, 15-20), and in some cases, base pair disruption occurs around the junction center (16, 20-22). Although the crystal structures of the resolvase-Holliday junction complexes have not been determined yet, the structures of RuvC (23), T4 endonuclease VII (13), T7 endonuclease I (11), and Hjc (12) suggest that these resolvases in common contain basic surfaces, which are most likely to contact the DNA. They all form homodimers related by 2-fold symmetry, which is suitable for introducing nicks into the two opposite strands of DNA, although their recognition schemes with the junction DNA seem to differ.

Hjc is a Holliday junction resolvase that is highly conserved within archaea. We originally identified it from the hyperthermophilic archaeon, Pyrococcus furiosus (24) and extensively characterized it (25-27). The crystal structure of P. furiosus Hjc revealed a positively charged surface containing highly conserved amino acid residues (12). Its folding is similar to that of the type II restriction endonucleases, including the conformation of the canonical catalytic residues as described above. The docking examination between Hjc and the junction DNA suggested that the N-terminal segment and the two loops participate in DNA binding, in good agreement with the results from mutational analyses (12). However, the actual roles of these regions in DNA recognition remain unclear.

To obtain detailed insights into the specific interaction between Hjc and Holliday junction DNA, we have determined the structure of a different crystal form and performed site-directed mutagenesis. The results indicate the unique and important roles of the flexible N-terminal segment in complex formation with the Holliday junction and its resolution.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Crystallization of the Hexagonal Crystal Form of Hjc-- The selenomethionine-containing Hjc was purified as described previously (12). The purified Hjc protein was stored in buffer containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 10% glycerol. The protein was concentrated by Centricon-10 ultrafiltration (Millipore) to 20 mg ml¹. Hjc was crystallized at 20 °C by the microbatch method with a silicone oil overlay, as reported previously (12). Good quality crystals were grown, when equal amounts of the protein solution and a solution containing 100 mM Tris-HCl, pH 7.0, 1 mM EDTA, 32.5% (w/v) polyethylene glycol 4000, and 10% glycerol were mixed. Hexagonal bipyramid-shaped crystals grew within 2 weeks. The crystal belongs to the space group P6₅, with unit cell dimensions a = b = 59.96 Å, c = 134.55 Å,  =  = 90°,  = 120°, and it contains two molecules of the Hjc protein in the asymmetric unit.

Data Collection and Molecular Replacement-- Crystals were harvested in harvesting buffer, containing 100 mM Tris-HCl, pH 7.0, 30% polyethylene glycol 4000, and 10% glycerol. X-ray diffraction data were collected at 100 K using a nitrogen stream. The diffraction data were collected at BL24XU beam line at SPring8 with a crystal to detector distance of 500 mm, using an R-AXIS V detector. These data were processed by MOSFLM (28). The scaling statistics are indicated in Table I. The Hjc dimer was used as a template for molecular replacement using CNS (29). All data between 41- and 2.16-Å resolution were included during simulated annealing with the bulk solvent correction. Ten percent of the reflections were kept separate to monitor the free R factor, and were not used in the refinement. A strict noncrystallographic symmetry restraint (300 kcal mol¹·Å²) and an overall B factor refinement were applied in the initial stage of the refinement. When the free R factor reached 0.32, the individual B factor was refined. After several rounds of manual rebuilding and refinement, solvent molecules were added in the refinement. The noncrystallographic symmetry restraint was released in the final two rounds of refinement. The final refinement statistics are shown in Table I. The current model contains regions of residues 1-114 for both molecules in the asymmetric unit.

Synthetic Holliday Junctions-- The synthetic Holliday junctions, 4Jh, 4Jb, and Z28, were prepared for the cleavage assay, the gel retardation assay, and the permanganate-probing experiments, respectively. The oligonucleotides used to make the junctions were described in previous papers (4Jh and 4Jb (25); Z28 (20)). For the BIACORE experiment, 4Jb-18 was labeled with biotin at the 5'-end.

Site-specific Mutagenesis-- A polymerase chain reaction-mediated mutagenesis was carried out using the Quick Change site-directed mutagenesis kit (Stratagene). The plasmid pFUHJ2 (24), for the expression of the hjc gene in Escherichia coli, was directly mutagenized according to the manufacturer's instructions with some modifications. The primers used for the R3A/K4A mutation were 5'- ATACATATGTATGCAGCAGGGGCCCAGGCA-3' and its complementary sequence. The resultant plasmid was designated as pHJ0304.

Purification of Wild-type and Mutant Hjc Proteins-- E. coli BL21 (DE3) carrying pFUHJ2, pHJM33, pDEL5, pHJ0304, pHJ3031, or pHJ5152, which contains the wild-type, D33A, 5, R3A/K4A, K30A/K31A, or K51A/K52A mutant hjc gene, respectively, was grown at 37 °C with shaking in 200 ml of L broth containing 20 mg of ampicillin. The induction of hjc gene expression and the purification of the mutant proteins were performed under the same conditions as described previously (27).

Cleavage and Binding Assay-- The Holliday junction cleavage assay using 4Jh and Z28 and the gel retardation assay using 4Jb were carried out as described (27).

Surface Plasmon Resonance (BIACORE) Analysis-- BIACORE 2000 and C1 sensor chips were from BIACORE Inc. Streptavidin was immobilized onto the sensor chip, which was utilized to capture the low concentration (about 100 response units) of a biotinylated synthetic Holliday junction, 4Jb. No target Holliday junction was captured on flow cell 1, which was used as the reference, while the other three flow cells were used to capture the Holliday junction. The assay was run at 20 °C in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 0.1 mg/ml bovine serum albumin, and 0.005% (v/v) Tween 20). The proteins were injected over the flow cells at concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 µM using a flow rate of 30 µl/min. All experiments included three injections of each protein concentration to determine the reproducibility of the signal and control injections to assess the stability of the surface during the experiment. The bound protein was removed with a 60-s wash with HBS buffer containing 2 M NaCl, which did not affect the affinity of Hjc to the immobilized Holliday junction (data not shown). The association and dissociation phase data were fit simultaneously using the nonlinear data analysis program, BIAevaluation 3.0 software (BIACORE). Binding data were described by a single-site interaction model including a term for mass transport of the protein to the sensor surface.

Modification of DNA by Potassium Permanganate-- Each of the wild-type or mutant Hjc proteins (1 µM) was incubated with 20 nM Holliday junction, Z28, with a 5'-³²P-labeled r strand, at 25 °C for 5 min in 20 µl of binding buffer (20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.1 mg/ml bovine serum albumin, 0.1 mM MgCl₂, and 50 µg/ml calf thymus DNA). Reactions were initiated by adding 2 µl of freshly dissolved 25 mM KMnO₄ and were stopped after 1 min by the addition of 1.5 µl of -mercaptoethanol. After ethanol precipitation, the DNA samples were reacted with 100 µl of 1 M piperidine for 30 min at 95 °C. Piperidine was removed by vacuum desiccation, and the pellets were washed three times with 50 µl of water and were vacuum-desiccated after each water addition. The pellets were resuspended in loading buffer (98% formamide, 10 mM EDTA, and 0.1% xylene cyanol FF) and were analyzed by electrophoresis on 12% denaturing polyacrylamide gels. The amounts of the thymine bases with the modification were quantified from autoradiograms using a laser-excited image analyzer (BAS5000; Fuji).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Crystal Structure of the Hexagonal Hjc-- A hexagonal crystal form of Hjc (hexagonal Hjc) was obtained under conditions similar to those used to obtain the monoclinic crystal form (monoclinic Hjc) reported previously (12) but without ammonium sulfate. The hexagonal Hjc crystal diffracted to a 2.16-Å resolution, and the structure was solved by molecular replacement. In the crystal, the Hjc resolvase adopts the same dimeric structure as observed in the monoclinic Hjc, and the overall architecture of each subunit is the same as that of the monoclinic Hjc (Fig. 1A). A notable exception is found in the N-terminal region, where the 1 helix in the hexagonal Hjc was shorter by one turn than in the monoclinic form (Fig. 1B). In the hexagonal Hjc, Gly⁵ at the end of the helix forms the 3-type turn (30), and thus the segment of four residues (Met¹-Lys⁴) extends to a 2-fold axis relating the dimer (Fig. 1C). It appears that this structural difference is due to the coordination of the sulfate ion with Arg³ and Lys⁴ in the monoclinic Hjc alone. Since this sulfate ion is bound to only one subunit of the Hjc dimer, the crystal packing may partly contribute to the formation of the extended helix. Nonetheless, this finding implies that the N-terminal segment of Hjc flexibly alters the conformation upon binding to DNA, in agreement with our previous proposal that the sulfate ion might mimic a phosphate moiety of Holliday junction DNA (12). This segment is located above the extensive basic surface formed by the N-terminal half of 1, the -sheet region facing to the N-terminal segment, and the N-terminal portion of 2. The basic surface, which is the putative interface with the junction DNA (12), contains a catalytic center similar to those of the type II restriction enzymes (Fig. 1A).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Structure of hexagonal Hjc. A, the Hjc dimer is shown in a stereoview. Each molecule of the dimer is colored differently. The active site residues are highlighted by the side chains. The N-terminal segment and the two loops are labeled and colored yellow. B, superposition of hexagonal (red) and monoclinic Hjc (blue). The sulfate bound to the monoclinic Hjc is highlighted. C, conformation of the N-terminal segment in the hexagonal Hjc. The refined model of the N-terminal segment is superimposed onto the corresponding electron density in the 2Fo  Fc simulated annealing omit map, which was calculated excluding the N-terminal five residues. The map was computed using all data between 41- and 2.16-Å resolution and was contoured at 0.5 .

Mutations at the Sulfate-binding Residues Decrease Junction Binding and Cleavage-- The residues of the sulfate-binding site are well conserved among different Hjc sequences (Fig. 2A). To determine whether the Hjc function is affected by a mutation in this region, we substituted alanine for Arg³ and Lys⁴. This mutant gene was expressed in E. coli, and the mutant Hjc protein (R3A/K4A) was purified to homogeneity (Fig. 2B). The junction cleavage assay indicated that the R3A/K4A mutations reduced the activity by 20-fold as compared with the wild-type Hjc (Fig. 2C). In addition, the binding of R3A/K4A to the Holliday junction was substantially lowered (Fig. 2D). We showed in a previous report that the truncation of five amino acids from the N terminus (5) causes a considerable decrease in the Hjc-Holliday junction complex formation and the cleavage activity (Ref. 12; see gel mobility shift assay in Fig. 2D). Taken together, these results indicate that the N-terminal segment of Hjc, including the two key residues, Arg³ and Lys⁴, is crucial to form a functional and stable complex with the Holliday junction.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effect of the R3A/K4A mutation on Holliday junction cleavage and binding activities of Hjc. A, a sequence alignment of the N-terminal regions is shown. Highly conserved residues are highlighted, and less conserved residues are gray. Pfu, P. furiosus; Pho, Pyrococcus horikoshii; Mja, Methanococcus jannaschii; Afu, Archaeoglobus fulgidus; Mth, Methanothermobacter thermoautotropicus; Ape, Aeropyrum pernix; Sso, Sulfolobus solfataricus. B, purified proteins (0.5 µg each) were analyzed by 15% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Brilliant Blue. B, Holliday junction cleavage assay for the R3A/K4A mutant Hjc. The wild-type (2, 10, or 100 nM) and R3A/K4A mutant proteins (2, 10, 100, or 500 nM or 1 µM) were incubated with 32P-labeled 4Jh (100 nM) at 60 °C for 30 min. After phenol extraction, the reaction products were analyzed by 6% polyacrylamide gel electrophoresis and were detected by autoradiography. Lane , no protein as a negative control. C, Holliday junction binding activity of the R3A/K4A mutant Hjc. Wild-type and mutant Hjc proteins (20, 50, and 200 nM) were incubated with 32P-labeled 4Jb (10 nM) on ice for 10 min. The products were analyzed by 6% polyacrylamide gel electrophoresis, followed by autoradiography.

Mutations in the N-terminal Segment Cause Faster Dissociation of the Complex-- The complex of the 5 mutant Hjc and Holliday junction was too unstable to be observed by a gel mobility shift assay unless they were cross-linked with glutaraldehyde (12). To measure the strength of the binding abilities of the wild-type and mutant Hjcs to Holliday junction precisely, the surface plasmon resonance technique was used. As shown in Fig. 3A, the wild-type Hjc showed binding specific to Holliday junction in a concentration-dependent manner. In this sensorgram, the association with the junction DNA occurred between 0 and 125 s, and the dissociation began after 125 s. The association (k_a) and dissociation (k_d) rate constants were evaluated to be 7.68 × 10⁴ M¹ s¹ and 1.14 × 10³ s¹, respectively, from the nonlinear curve fitting of the sensorgram (Fig. 3A and Table II). The affinity was calculated from the equilibrium dissociation constant (K_D = k_d/k_a) to be 14.9 nM. This value is consistent with the apparent value of 60 nM previously obtained from the gel mobility shift assay (25). The minor discrepancy in these values may be attributed to the different methods. We used this procedure to measure the k_a and k_d values of the mutant Hjcs (Fig. 3, B-F, and Table II). The apparent K_D values were similar between the various mutants, whereas the individual k_a and k_d values were strikingly different (Table II). The D33A mutant, with impaired catalytic activity (26, 27), showed k_a and k_d values similar to those of the wild-type Hjc (Fig. 3B and Table II). On the other hand, the other four Hjc mutants showed significant increases in both the association and dissociation rates for the junction binding, as revealed from their calculated k_a and k_d values. In particular, the 5 and R3A/K4A mutants showed the largest increases, especially in k_d (Fig. 3, C and D, and Table II), indicating that the Hjc-Holliday junction complexes formed with 5 and R3A/K4A are very unstable. These results reveal that the N-terminal segment plays a crucial role in stabilizing the complex. The K30A/K31A and K51A/K52A mutants (Fig. 3, E and F, and Table II), where the adjacent two lysines were replaced by alanines, showed a similar mild increase in both the k_a and k_d values. These results are consistent with the observations in our previous gel mobility shift assay (12).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Analysis of the interaction between Hjc and Holliday junction using the surface plasmon resonance system. The analysis of the Hjc-Holliday junction interaction is described in detail under "Experimental Procedures." The sensorgram was obtained at 20 °C and pH 7.4. Five different concentration series were used in a single experiment to obtain the association and dissociation rate constants. Diamonds, 0.5 µM; reverse triangles, 0.4 µM; triangles, 0.3 µM; squares, 0.2 µM; circles, 0.1 µM. The results of the simultaneous nonlinear curve fit are shown as a thick line. A, wild-type Hjc; B, D33A Hjc; C, 5 Hjc; D, R3A/K3A Hjc; E, K30A/K31A Hjc; F, K51A/K52A Hjc.

The N-terminal Segment Is Involved in a Local Base Pair Disruption-- It has been shown that Sulfolobus solfataricus Hjc disrupts the base pairs around the crossover point (20). To clarify this mechanism, we have analyzed base pairing of junction DNA bound to the mutant Hjcs as well as to the wild-type Hjc by a potassium permanganate probing technique (Fig. 4). Although we used Holliday junction with the same sequence as that used for the S. solfataricus Hjc, a notable difference was observed in the cleavage pattern. In the case of the S. solfataricus Hjc, a biased cleavage occurred in strands h and x, whereas in the P. furiosus Hjc, each strand was cleaved with an almost equivalent preference (Fig. 4, A (lanes 2, 4, 6, and 8) and B). In the probing assay, this cleavage was observed as a band in the absence of potassium permanganate (Fig. 4C, lane 3), and several other bands derived from modified thymines came up in the presence of potassium permanganate (Fig. 4C, lane 4). These results indicate that P. furiosus Hjc also unstacks the DNA junction and possibly disrupts the base pairs at the junction point upon binding. Due to its own cleavage activity, the wild-type Hjc showed the bands from the modified thymines with much weaker intensity than those in the case of the D33A mutant Hjc (Fig. 4C, lanes 4 and 5). Among the mutant proteins, R3A/K4A (Fig. 4C, lane 7) and K30A/K31A (Fig. 4C, lane 8) had little or almost no cleavage activity and showed results similar to that for D33A, while K51A/K52A, which retained a weak cleavage activity, exhibited binding activity similar to that of the wild-type Hjc (Fig. 4C, lane 9). The sensitivities for cleavage versus base pair positions in Holliday junction are plotted in Fig. 4D. These results indicate that the D33A, R3A/K4A, K30A/K31A, and K51A/K52A mutants retain proper binding ability to the Holliday junction. In contrast, the 5 mutant did not show the strong bands derived from the potassium permanganate-sensitive thymines, although the mutant showed the same k_a and k_d values as those of R3A/K4A (Fig. 4C, lane 6). This result implies that the N-terminal segment, but not the particular Arg³ and Lys⁴ residues, is responsible for the base unstacking and the disruption of the base pairs at the junction center.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Base unstacking probed by potassium permanganate. A, the synthetic Holliday junctions, Z28 (20 nM), each uniquely labeled with 32P at the 5'-end of the indicated strand, were incubated with or without Hjc protein (100 nM) at 60 °C for 10 min. The reaction products after phenol extraction were separated on a 12% denaturing polyacrylamide gel and were detected by autoradiography. B, the central sequence of the Z28 junction. The four thymine bases are located on either side of the branch point of the r strand. The arrowheads indicate the cleavage sites generated by Hjc. C, the Z28 junction, labeled with 32P on the r strand, was incubated with the indicated mutant Hjc proteins and was reacted with potassium permanganate as described under "Experimental Procedures." The white and black arrowheads indicate the junction branch point and the cleavage product band generated by Hjc, respectively. Lane GA, A + G markers of strand r. D, the enhancement in reactivity to permanganate was calculated for each of the eight thymine positions in the r strand of Z28, and the averages from the results of duplicate experiments are plotted. Open squares, wild-type Hjc; closed squares, D33A; open circles, 5; closed circles, R3A/K4A; open triangles, K30A/K31A; closed triangles K51A/K52A mutant Hjc.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the previous report on the monoclinic Hjc structure, we proposed that the sulfate ion bound at the N-terminal region might reflect a phosphate moiety of the backbone of the junction DNA. The mutant Hjc lacking the N-terminal five amino acids showed a drastic decrease in the stability of the complex with the Holliday junction, and thus we concluded that this region is important for DNA recognition (12). The present mutational analyses of the sulfate binding residues, Arg³ and Lys⁴, confirmed that these residues are indeed critical for the formation of a stable complex with the junction DNA but are not essential for the cleavage activity. Alterations of these residues (R3A/K4A) yielded a significant increase in the dissociation rate of the Hjc-Holliday junction complex. Furthermore, the R3A/K4A mutant was able to unstack the junction DNA and disrupt the base pairs when it bound the substrate. In contrast, the truncation mutant 5, which has the same k_a and k_d values as those of R3A/K4A for Holliday junction binding, can neither unstack the junction DNA nor cleave the DNA. These results imply that the N-terminal segment plays two major roles: stabilization of the complex and correct positioning of the DNA for cleavage.

The different conformations of the N-terminal segment in the crystal structure, in the absence or presence of sulfate, suggested that it is more flexible than other parts of the Hjc protein. This conformational flexibility may be favorable for Hjc to find and to fit the DNA conformation that is suitable for cleavage. The multiple sequence alignment shows that Gly⁵ is absolutely conserved among the different Hjc sequences (Fig. 2A). Since glycine is a key residue for the versatility of peptide conformations, this conformational flexibility may be an inherent property of Hjc.

The two sets of contiguous lysines, Lys³⁰/Lys³¹ and Lys⁵¹/Lys⁵², which lie in two different loops, contribute to the interaction with the Holliday junction. Both of them appear to play similar roles in stabilizing the complex with the Holliday junction. However, they are considerably different in terms of the cleavage activity; the K51A/K52A mutant retains the cleavage activity with less efficiency than the wild-type Hjc, whereas the K30A/K31A mutant could not cleave Holliday junction (12). It is possible that Lys³⁰ and Lys³¹ in the loop play an additional role in arranging the DNA into an appropriate position for cleavage. Our previous analysis revealed that several conserved basic residues (Arg¹⁰, Arg²⁵, and Lys⁸¹) are important for the cleavage activity but not for the DNA binding (27). These residues are also located on the basic surface, and hence they may have a function similar to that shared by Lys³⁰ and Lys³¹.

The combined approach of mutational and structural analyses has allowed us to propose the following scheme for Holliday junction recognition and cleavage by Hjc. First, the junction DNA encounters the extensive basic surface of Hjc. Then the flexible N-terminal segment plays a major role in stabilizing the Hjc-Holliday junction complex together with the secondary contribution by the two sets of lysines, Lys³⁰/Lys³¹ and Lys⁵¹/Lys⁵². Next, the DNA is arranged in the appropriate position relative to the catalytic residues of Hjc for incision. This step involves two modes of interactions between the protein and DNA. One is the disruption of the base pairs around the junction center, in which the N-terminal segment plays a major part. The other is the interaction of basic residues with DNA, involving the Lys³⁰/Lys³¹ loop and several basic residues (Arg¹⁰, Arg²⁵, and Lys⁸¹). After correct positioning, the phosphodiester bonds are cleaved in the nuclease center, composed of the canonical catalytic residues (Glu⁹, Asp³³, Glu⁴⁶, and Lys⁴⁸) (27). Base pair disruption is observed in a number of complexes of Holliday junction and the binding proteins; the crystal structure of the E. coli RuvA-Holliday junction complex revealed unpaired bases at the junction center (31). Yeast CceI and E. coli RuvC also distort base pairs around the crossover point (15, 21), and in particular, the Phe⁶⁹ side chain in RuvC appears to make a stacking interaction with the base pairs near the junction center (22). S. solfataricus Hjc also disrupts base pairs (20), and as shown by the present study, the N-terminal segment appears to play a major role in this process. Previous modeling of the Hjc-Holliday junction complex has indicated that this segment is close to a phosphate backbone and might interact with the DNA minor groove (12). Moreover, the present crystal structure has highlighted the importance of the conformational flexibility of N-terminal segment, in both the induced fit mechanism and the correctly positioning of the segment close to the junction center. The disruption mechanism should be solved to prove our hypothesis, although it is possible that the base pair disruption is caused by simple tension resulting from the interaction between the peptide groups of the N-terminal segment and the DNA. The primary sequence of the N-terminal segment contains an aromatic residue, but it is not well conserved (Fig. 2A). Furthermore, since we have expressed recombinant Hjc in E. coli, there may be some modification within this region in the native form. However, our biochemical analyses suggested that both the Holliday junction binding and cleavage activity are similar between the native and the recombinant Hjcs (24). Therefore, we presume that drastic modification would not be taking place in the P. furiosus cells.

During our manuscript preparation, the crystal structure of Hjc from S. solfataricus was reported (32). The structure was similar to that of P. furiosus Hjc with a root mean square displacement value of 2.4 Å for residues 6-114. Interestingly, in S. solfataricus Hjc, two regions, the N-terminal segment and Lys³⁰/Lys³¹ loop, were structurally disordered. The flexibility of the N-terminal segment agrees well with our data for P. furiosus Hjc. In addition, the flexibility of the Lys³⁰/Lys³¹ loop is intriguing, because our mutational and biochemical analyses suggest its involvement in DNA recognition and cleavage.

In conclusion, our structural and mutational analyses of Hjc have revealed the individual roles of various regions, which are important for the recognition and cleavage of Holliday junction. In particular, the flexible N-terminal segment greatly enhances the stability of the enzyme-junction DNA complex and contributes to the correct positioning of the cleavage site of the DNA to the catalytic site of Hjc. The present data have provided clearer insights into the tertiary structural view of the Hjc-Holliday junction complex. The elucidation of the precise recognition mechanism must await the Hjc-Holliday junction complex structure at atomic resolution.

	ACKNOWLEDGEMENTS

We thank Yoshio Katsuya and Naoki Kunishima for help in the use of the SPring8 BL24XU.

	FOOTNOTES

^* 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.

The atomic coordinates and the structure factors (code 1IPI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Research Fellow of the Japan Society for the Promotion of Science.

^¶ To whom correspondence should be addressed. Tel.: 81-6-6872-8201; Fax: 81-6-6872-8219; E-mail: morikawa@beri.co.jp.

Published, JBC Papers in Press, July 5, 2001, DOI 10.1074/jbc.M104460200

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

1.	Holliday, R. (1964) Genet. Res. 5, 282-304
2.	White, M. F., Giraud-Panis, M. J., Pohler, J. R., and Lilley, D. M. (1997) J. Mol. Biol. 269, 647-664
3.	Kemper, B. (ed) (1997) in DNA Damage and Repair Biochemistry, Genetics, and Cell Biology (Nickoloff, J. A. , and Hoekstra, M., eds) , pp. 179-204, Humana Press, Totowa, NJ
4.	Sharples, G. J. (2001) Mol. Microbiol. 39, 823-834
5.	Hyde, H., Davies, A. A., Benson, F. E., and West, S. C. (1994) J. Biol. Chem. 269, 5202-5209
6.	Constantinou, A., Davies, A. A., and West, S. C. (2001) Cell 104, 259-268
7.	Aravind, L., Makarova, K. S., and Koonin, E. V. (2000) Nucleic Acids Res. 28, 3417-3432
8.	Lilley, D. M., and White, M. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9351-9353
9.	Mizuuchi, K. (1997) Genes Cells 2, 1-12
10.	Morikawa, K. (1998) in Nucleic Acids and Molecular Biology (Eckstein, F. , and Lilley, D. M. J., eds) , pp. 275-299, Springer-Verlag, Berlin
11.	Hadden, J. M., Convery, M. A., Declais, A. C., Lilley, D. M., and Phillips, S. E. (2001) Nat. Struct. Biol. 8, 62-67
12.	Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K. (2001) Structure 9, 197-204
13.	Raaijimakers, H., Vix, O., Toro, I., Golz, S., Kemper, B., and Suck, D. (1999) EMBO J. 18, 1447-1458
14.	Duckett, D. R., Murchie, A. I., Bhattacharyya, A., Clegg, R. M., Diekmann, S., von Kitzing, E., and Lilley, D. M. (1993) Eur. J. Biochem. 211, 285-295
15.	Bennett, R. J., and West, S. C. (1995) J. Mol. Biol. 252, 213-226
16.	Duckett, D. R., Panis, M. J., and Lilley, D. M. (1995) J. Mol. Biol. 246, 95-107
17.	Pohler, J. R., Giraud-Panis, M. J., and Lilley, D. M. (1996) J. Mol. Biol. 260, 678-696
18.	Chan, S. N., Vincent, S. D., and Lloyd, R. G. (1998) Nucleic Acids Res. 26, 1560-1566
19.	Giraud-Panis, M. J., and Lilley, D. M. (1998) J. Mol. Biol. 278, 117-133
20.	Kvaratskhelia, M., Wardleworth, B. N., Norman, D. G., and White, M. F. (2000) J. Biol. Chem. 275, 25540-25546
21.	White, M. F., and Lilley, D. M. (1997) J. Mol. Biol. 266, 122-134
22.	Yoshikawa, M., Iwasaki, H., and Shinagawa, H. (2001) J. Biol. Chem. 276, 10432-10436
23.	Ariyoshi, M., Vassylyev, D. G., Iwasaki, H., Nakamura, H., Shinagawa, H., and Morikawa, K. (1994) Cell 78, 1063-1072
24.	Komori, K., Sakae, S., Shinagawa, H., Morikawa, K., and Ishino, Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8873-8878
25.	Komori, K., Sakae, S., Fujikane, R., Morikawa, K., Shinagawa, H., and Ishino, Y. (2000) Nucleic Acids Res. 28, 4544-4551
26.	Daiyasu, H., Komori, K., Sakae, S., Ishino, Y., and Toh, H. (2000) Nucleic Acids Res 28, 4540-4543
27.	Komori, K., Sakae, S., Daiyasu, H., Toh, H., Morikawa, K., Shinagawa, H., and Ishino, Y. (2000) J. Biol. Chem. 275, 40385-40391
28.	Leslie, A. G. W. (1992) Joint CCP4 and ESF-EACM Newsletter on Protein Crystallography, No. 26, SERC Daresbury Laboratory, Warrington, UK
29.	Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921
30.	Kabsch, W., and Sander, C. (1983) Biopolymers 22, 2577-2637
31.	Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H., and Morikawa, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8257-8262
32.	Bond, C. S., Kvaratskhelia, M., Richard, D., White, M. F., and Hunter, W. N. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5509-5514

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