A Conserved Nuclease Domain in the Archaeal Holliday Junction Resolving Enzyme Hjc

Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3′ to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme. Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3′ to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme. base pairs Holliday junction resolving enzymes play a role in the pathway of homologous recombination, recognizing and cleaving the four-way DNA junctions that arise from strand exchange between homologous duplex DNA species. Junction resolving enzymes are ubiquitous in nature. These proteins have been identified in Eubacteria (RuvC (1Iwasaki H. Takahagi M. Shiba T. Nakata A. Shinagawa H. EMBO J. 1991; 10: 4381-4389Crossref PubMed Scopus (223) Google Scholar, 2Connolly B. Parsons C.A. Benson F.E. Dunderdale H.J. Sharples G.J. Lloyd R.G. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6063-6067Crossref PubMed Scopus (150) Google Scholar) and RusA (3Mahdi A.A. Sharples G.J. Mandal T.N. Lloyd R.G. J. Mol. Biol. 1996; 257: 561-573Crossref PubMed Scopus (128) Google Scholar)), bacteriophage (T4 endonuclease VII (4Mizuuchi K. Kemper B. Hays J. Weisberg R.A. Cell. 1982; 29: 357-365Abstract Full Text PDF PubMed Scopus (185) Google Scholar) and T7 endonuclease I (5Dickie P. McFadden G. Morgan A.R. J. Biol. Chem. 1987; 262: 14826-14836Abstract Full Text PDF PubMed Google Scholar, 6de Massey B. Weisberg R.A. Studier F.W. J. Mol. Biol. 1987; 193: 359-376Crossref PubMed Scopus (90) Google Scholar)), fungal mitochondria (Cce1 (7Kleff S. Kemper B. Sternglanz R. EMBO J. 1992; 11: 699-704Crossref PubMed Scopus (125) Google Scholar)), and most recently Archaea (Hjc and Hje (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar, 9Komori K. Sakae S. Shinagawa H. Morikawa K. Ishino Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8873-8878Crossref PubMed Scopus (91) Google Scholar)). Whereas activities have been detected in nuclear extracts from yeast (10West S.C. Parsons C.A. Picksley S.M. J. Biol. Chem. 1987; 262: 12752-12758Abstract Full Text PDF PubMed Google Scholar) and mammalian cells (11Hyde H. Davies A.A. Benson F.E. West S.C. J. Biol. Chem. 1994; 269: 5202-5209Abstract Full Text PDF PubMed Google Scholar, 12Elborough K.M. West S.C. EMBO J. 1990; 9: 2931-2936Crossref PubMed Scopus (87) Google Scholar), the relevant genes have yet to be identified. Resolving enzymes function as dimers, resolving the four-way DNA junction by the introduction of paired nicks in opposing strands with a magnesium-dependent endonuclease activity. Despite these functional similarities, the junction resolving enzymes are structurally diverse with no detectable sequence similarity among any of the known examples. Structural studies have highlighted this diversity because the crystal structures of RuvC (13Ariyoshi M. Vassylyev D.G. Iwasaki H. Nakamura H. Shinagawa H. Morikawa K. Cell. 1994; 78: 1063-1072Abstract Full Text PDF PubMed Scopus (264) Google Scholar), T4 endonuclease VII (14Raaijmakers H. Vix O. Toro I. Golz S. Kemper B. Suck D. EMBO J. 1999; 18: 1447-1458Crossref PubMed Scopus (111) Google Scholar), and T7 endonuclease I 1S. E. V. Phillips and D. M. J. Lilley, personal communication.1S. E. V. Phillips and D. M. J. Lilley, personal communication. have radically different folds. These observations have led to the suggestion that resolving enzymes have arisen several times during the course of evolution, perhaps by recruitment of nucleases with other cellular roles. This is almost certainly the case for the eubacterial enzyme RuvC, which shares a fold and metal binding site with members of the RNase HI superfamily (13Ariyoshi M. Vassylyev D.G. Iwasaki H. Nakamura H. Shinagawa H. Morikawa K. Cell. 1994; 78: 1063-1072Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 15Saito A. Iwasaki H. Ariyoshi M. Morikawa K. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7470-7474Crossref PubMed Scopus (83) Google Scholar). The Archaea constitute a third domain of life that is distinct from both the Eubacteria and the Eucarya. Whereas the Archaea resemble their fellow prokaryotes in most respects, they share many similarities with the Eucarya in the information processing pathways including DNA replication, transcription, and translation (reviewed in Ref. 16Keeling P.J. Doolittle W.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5761-5764Crossref PubMed Scopus (90) Google Scholar), and the archaeal processes constitute a useful model system for the much more complex eucaryal equivalents. We are investigating the pathway of homologous recombination in the Archaea and have detected two Holliday junction resolving enzymes, Hje and Hjc, in the CrenarchaeoteSulfolobus solfataricus (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar, 17Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 295: 193-202Crossref PubMed Scopus (44) Google Scholar). The gene for Hjc has been identified and is conserved in all Archaea for which extensive genome sequence is available (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar, 9Komori K. Sakae S. Shinagawa H. Morikawa K. Ishino Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8873-8878Crossref PubMed Scopus (91) Google Scholar). We have cloned S. solfataricusHjc and overexpressed the recombinant enzyme in Escherichia coli (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar). Here we demonstrate manipulation of the global structure of the four-way junction substrate by Hjc and disruption of base stacking near the cleavage site. We report the identification of a structural motif in Hjc that is shared by a diverse group of nucleases and constitutes a binding site for the catalytic metal ions. Parallels between Holliday junction resolving enzymes and restriction enzymes that were first pointed out at a biochemical level for the yeast mitochondrial enzyme Cce1 (18Schofield M.J. Lilley D.M.J. White M.F. Biochemistry. 1998; 37: 7733-7740Crossref PubMed Scopus (42) Google Scholar) now achieve a firm structural basis in the archaeal Hjc enzyme. Oligonucleotides were synthesized and four-way DNA junctions assembled as described previously (18Schofield M.J. Lilley D.M.J. White M.F. Biochemistry. 1998; 37: 7733-7740Crossref PubMed Scopus (42) Google Scholar) using the sequences described in the following paragraphs. This fixed four-way junction was prepared with arms of 15 or 25 bp2 as described previously (45Duckett D.R. Murchie A.I.H. Diekmann S. von Kitzing E. Kemper B. Lilley D.M.J. Cell. 1988; 55: 79-89Abstract Full Text PDF PubMed Scopus (417) Google Scholar). Comparative gel electrophoresis experiments utilized a version of junction 3 with four arms of 60 bp in length. Six forms of junction 3 with two long and two short arms were derived from this junction as described previously (46Pöhler J.R.G. Giraud-Panis M.-J.E. Lilley D.M.J. J. Mol. Biol. 1996; 260: 678-696Crossref PubMed Scopus (80) Google Scholar). This is a fixed junction with 20 bp in each arm assembled from four oligonucleotides, each of 40 nucleotides in length as described previously (47White M.F. Lilley D.M.J. J. Mol. Biol. 1996; 257: 330-341Crossref PubMed Scopus (89) Google Scholar). This junction is utilized for permanganate-probing experiments. The junction has arms of 15 bp including eight thymine residues centered around the point of strand exchange on the r strand: b, 5′-TCCGTCCTAGCAAGGAGTCTGCTACCGGAA; h, 5′-TTCCGGTAGCAGACTAAAAGGTGGTTGAAT; r, 5′-ATTCAACCACCTTTTTTTTAACTGCAGCAG; x, 5′-CTGCTGCAGTTAAAACCTTGCTAGGACGGA. Recombinant Hjc protein was expressed in E. colistrain BL21 (DE3) CodonPlus RIL (Stratagene), and the protein was purified as described previously (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar). In brief, Hjc was purified by chromatography on an SP-Sepharose high performance 26/10 column (Hi-Load, Amersham Pharmacia Biotech) equilibrated with Buffer A (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol). A 500-ml linear gradient of 0–1000 mm NaCl was used to elute cationic proteins. Fractions corresponding to a distinct absorbance peak were analyzed by SDS-polyacrylamide gel electrophoresis, pooled, concentrated, and loaded onto a 26/70 gel filtration column (Superdex 200 Hi-Load, Amersham Pharmacia Biotech) and developed with Buffer A containing 300 mm NaCl. Active fractions were pooled and shown to contain essentially homogeneous Hjc protein. This protein was used for all subsequent analyses. Site-directed mutagenesis of Hjc was carried out in the plasmid pUC119 using the QuikChange method (Stratagene). After mutagenesis, DNA sequencing was used to confirm that no spurious mutations had been introduced. The Hjc mutants were subcloned into pET19b and were expressed and purified similar to the wild-type enzyme. The oligonucleotides used to introduce the mutations are listed as follows: E12Qfor, 5′-GGAAAGGTTCCGCAGTACAACGAAATATTGTGAG; E12Qrev, 5′-CTCACAATATTTCGTTGTACTGCGGAACCTTTCC; D42Nfor, 5′-GACCCTATACCGAATATTATCGCT; D42Nrev, 5′-AGCGATAATATTCGGTATAGGGTC; E55Qfor, 5′-CGTTATTATTTTAATTCAGATGAAGAGTAG; E55Qrev, 5′-CTACTCTTCATCTGAATTAAAATAATAACG; K57Afor, 5′-TTTTAATTGAGATGGCGAGTAGAAAGG; K57Arev, 5′-CCTTTCTACTCGCCATCTCAATTAAAA. Samples of purified Hjc protein (1 μm) were incubated with radioactive 5′-32P-labeled four-way DNA junction 3 in binding buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.1 mg/ml bovine serum albumin, and 0.2 mg/ml calf thymus duplex competitor DNA) including either 1 mm EDTA or 0.1 mm MgCl2 in a 10-μl total volume for 5 min at 20 °C, prior to addition of loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 15% Ficoll type 400) at a dilution of 1:6 (v/v). Samples were loaded onto 5% polyacrylamide gels and electrophoresed in Tris-borate-EDTA buffer or Tris-borate and 0.1 mm MgCl2 with buffer recirculation. After electrophoresis, gels were dried on Whatman 3MM paper and exposed to x-ray film for documentation. Binding affinity was measured by gel electrophoretic retardation analysis using radioactive 5′-32P-labeled junction 1 in EDTA binding buffer as described previously (8Kvaratskhelia M. White M.F. J. Mol. Biol. 2000; 297: 923-932Crossref PubMed Scopus (47) Google Scholar). Assays were carried out using 1 μm purified recombinant Hjc protein in reaction buffer (20 mm Tris-HCl, pH 7.5, 50 mm NaCl, 15 mm MgCl2) using 80 nm 5′-32P-labeled junction as a substrate. Calf thymus DNA (0.2 mg/ml) was added as a competitor to minimize nonspecific endonuclease activity and DNA-binding proteins. Reactions were initiated by the addition of magnesium to the assay mix in a 5-μl total volume and incubated at 60 °C. At set time points aliquots were removed, and the reactions were stopped by the addition of 4 μl of formamide/EDTA loading mix with heating to 95 °C. Products were analyzed by denaturing gel electrophoresis and phosphor imaging as described previously (48White M.F. Lilley D.M.J. Mol. Cell. Biol. 1997; 17: 6465-6471Crossref PubMed Scopus (54) Google Scholar). Radioactive 5′-32P-labeled four-way DNA junction Z28 was incubated at room temperature for 5 min in the presence or absence of 1 μm Hjc protein in binding buffer. The total reaction volume was 20 μl. Reactions were initiated by the addition of 2 μl of freshly dissolved 25 mm KMnO4 and were stopped after 1 min with the addition of 1.5 μl of β-mercaptoethanol. After ethanol precipitation, 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 30 μl of water and were vacuum desiccated after each water addition. The dried samples were resuspended in formamide loading mix and analyzed on 15% denaturing polyacrylamide gels. The Holliday junction assumes a conformation known as the “stacked X structure” in the presence of divalent metal ions, folding by coaxial stacking of pairs of helices in an antiparallel 2-fold symmetric cross (reviewed in Ref. 19Lilley D.M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9513-9515Crossref PubMed Scopus (29) Google Scholar). This structure has been studied by a wide variety of techniques and has recently been confirmed by x-ray crystallography (20Lilley D.M. Norman D.G. Nat. Struct. Biol. 1999; 6: 897-899Crossref PubMed Scopus (20) Google Scholar, 21Ortiz-Lombardia M. Gonzalez A. Eritja R. Aymam J. Azorin F. Coll M. Nat. Struct. Biol. 1999; 6: 913-917Crossref PubMed Scopus (177) Google Scholar). Comparative gel electrophoresis has proven a powerful technique for the analysis of the global configuration of the four-way junction, both in solution and complexed with proteins. Using this technique, all Holliday junction binding proteins studied to date have been shown to alter the conformation of the Holliday junction on binding (reviewed in Ref. 22White M.F. Giraud-Panis M.-J.E. J.R.G. Lilley D.M.J. J. Mol. Biol. 1997; 269: PubMed Scopus Google Scholar). We the of Hjc binding on the global conformation of the four-way junction using gel In EDTA, the junction the of the symmetric junction with arms by In the this was with a the was in the presence of 0.1 mm the junction a as previously for which with X and stacking A.I.H. J. Lilley D.M.J. EMBO J. 1991; 10: PubMed Scopus Google Scholar). the the These observations that the four-way DNA junction is by Hjc in a 2-fold symmetric conformation with the arms and arms by and the and pairs by This X is of that on by the resolving enzyme RuvC, in that case the structure from and between the and pairs West S.C. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). Whereas we the between the pairs of the in of the that the from the for Cce1 and and or a for the and Using a we measured the of cleavage of each of the four strands of junction was to be with a for the and r strands with the and strands this to the global structure of in complex with Hjc, the more strands are in the strands with 3 nucleotides 3′ of the point 1 and in the other two strands a of the junction that has been by Hjc in the other conformation with the and pairs of arms by residues in DNA are to by are from in the of duplex DNA. This the to cleavage by by is a for base stacking in duplex DNA. Holliday junction binding proteins as Cce1 and the of at the point to in the presence of magnesium ions, and this has been as that the proteins the DNA junction and base pairing at the point of strand exchange West S.C. J. Mol. Biol. 1995; PubMed Scopus Google M.F. Lilley D.M.J. J. Mol. Biol. 1997; PubMed Scopus (83) Google Scholar). was used to base stacking in the of the complex with junction which has eight the point of strand exchange on the r In the absence of Hjc all eight were to by with the at the 2 nucleotides the point of strand exchange and with to the junction The presence of magnesium in the reaction the of the to with junction stacking these 3 In the presence of of the Hjc we a in with in both EDTA and magnesium 4 and of the in for each of the thymine residues to a in the the of the junction the protein the duplex DNA in this of the cleavage of junction Z28 by Hjc reveals similar to junction cleavage is in the and strands 3 nucleotides 3′ of the point The of cleavage of the strand with the of detected on the r that Hjc introduce structural in the DNA near the cleavage site. The base pairing in this region of the DNA duplex be or by Hjc on are of DNA by restriction enzymes as D. K. EMBO J. PubMed Scopus Google Scholar) and C.A. P. J. PubMed Scopus Google Scholar). of the residues conserved in a of all known Hjc sequences a motif containing three and by similar to a conserved motif in type restriction enzymes Struct. Biol. Scopus Google Scholar) This motif constitutes of the site of the restriction enzymes, with the three residues the metal binding for the two catalytic magnesium D. Biochemistry. 1995; PubMed Scopus Google Scholar) and the conserved to play a role in the during D. K. EMBO J. PubMed Scopus Google Scholar). the secondary structure of Hjc in this we a of Hjc proteins to the structure B. J. Mol. Biol. PubMed Scopus Google Scholar). The are in 4 with the conserved domain of Hjc with the corresponding of The secondary structure for Hjc of an containing the first by three in with the known secondary structures of the three restriction enzymes V. Nat. Struct. Biol. 1995; PubMed Scopus Google Scholar). model for the metal binding site of the Hjc we site-directed mutants of the three conserved residues and the conserved The proteins were expressed in E. coli and purified as for the wild-type enzyme. The was expressed and of protein be The other three mutants were expressed at similar to the wild-type protein and the to to the four-way junction with an affinity with the wild-type Hjc protein 5 the catalytic activities of the wild-type and enzymes, we incubated the proteins with the four-way junction substrate Z28 in cleavage buffer at 60 and cleavage from substrate by denaturing gel electrophoresis 5 A activity in the a in activity with the wild-type enzyme. and no detectable catalytic activity. four of these mutations to have had a on on substrate with the functional by to the residues and other Hjc a motif first identified as of the site of the type restriction enzyme crystal structures of the restriction A. M. D. M. Nat. Struct. Biol. 1994; PubMed Scopus Google Scholar) and M. K. G. J. I. Phillips EMBO J. 1998; 17: PubMed Scopus Google Scholar) that the metal binding is structurally conserved in an catalytic domain in the three enzymes, the are The type restriction enzymes and this domain (reviewed in R.A. Chem. Biol. 1999; PubMed Scopus Google and EMBO J. 1998; 17: PubMed Scopus (177) Google Scholar), and more recently has been identified in EMBO J. 1998; 17: PubMed Scopus (177) Google Scholar), R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), and type I and endonucleases I. A. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). of these enzymes have a catalytic domain in the of an with different that and DNA EMBO J. 1998; 17: PubMed Scopus (177) Google Scholar, A. V. 1994; PubMed Scopus Google Scholar). The structures and of these proteins are restriction enzymes are and and is and activity is by the and proteins following DNA EMBO J. 1998; 17: PubMed Scopus (177) Google Scholar). a that strand of a DNA duplex after the of a strand has a structure R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the most example of the of and is in the restriction which has the catalytic domain from the DNA domain by a I. 1997; PubMed Scopus Google Scholar). has utilized a catalytic metal ion binding domain in with an of DNA and protein to many diverse in We have shown by site-directed mutagenesis that mutations in residues and of Hjc all in at a in catalytic the mutants the to the four-way mutagenesis of the residues in in similar in catalytic activity substrate binding A. H. U. E. H. A. Biochemistry. 1995; PubMed Scopus Google Scholar, U. T. E. M. V. J. T. H. F. A. Biochemistry. 1992; PubMed Scopus Google Scholar, Biochemistry. 1996; PubMed Scopus Google Scholar, J. Mol. Biol. 1999; PubMed Scopus (44) Google Scholar, A. U. A. Biochemistry. 1997; PubMed Scopus Google Scholar). This is the first example of this domain in a Holliday junction resolving enzyme and the first in an archaeal protein of known Using the sequence shown in we used the crystal structure of the metal binding domain of to a model of the catalytic domain of Hjc The model the of the Hjc protein of an and a antiparallel The three residues group in to as for two magnesium in the and the of the conserved is with a role in with the other nucleases containing the metal ion binding the of the which is conserved in Hjc proteins from different archaeal play a role in DNA a model for the complex of the catalytic domain of Hjc, on the of a four-way DNA junction, which is in the X conformation by gel The catalytic magnesium ion has been within 5 of the known site of cleavage on the Using this the model a of binding of the Hjc protein with the four-way DNA junction substrate. residues in Hjc are in to the and in the model roles for two and These are the residues other the catalytic that are conserved in all Hjc and in the model of the complex they in to with on an arm of the the basis of the model and the of these residues we that they are for DNA binding and for of the branched structure of the four-way DNA In we have that the archaeal Holliday junction resolving enzyme Hjc manipulates the global structure of the four-way DNA junction into a 2-fold symmetric X conformation on nicks in the are on the in a region of duplex DNA 3 bp from the point of strand by local of base stacking in the Hjc an domain for the in the of four-way DNA with four conserved catalytic residues in many other nucleases of diverse The of the Hjc protein the domain and an for the Hjc of two of nicks in duplex DNA on either of the point of a four-way junction, with other structural structural specificity and the We for out the sequence similarity between Hjc and the catalytic domain of the restriction enzymes.