Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance Barbara Sigala and Irina R. Tsaneva Department of Biochemistry and Molecular Biology, University College London, London, UK The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant struc- tural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the a-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the trun- cated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601–6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Dydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA ÔphenotypeÕ. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe. Keywords: Holliday junction resolvase; mtDNA; yeast. The Holliday junction is a key intermediate in homologous recombination and double-strand break repair pathways that proceeds via the reciprocal exchange of strands between homologous DNA duplexes. Holliday junctions could also arise from the regression of stalled replication forks [1–3] and are thought to play an important role in the repair and restart of stalled replication forks (reviewed in [1]). The correct processing of this crossover intermediate is therefore crucial for the integrity and maintenance of DNA in all organisms including mitochondrial DNA (mtDNA). Mitochondrial DNA amounts to about 15% of the DNA content in Saccharomyces cerevisiae. A haploid cell contains about 50 copies of the 75 kb mitochondrial genome as clusters of linear concatamers (reviewed in [4]). Recombi- nation between mtDNA genomes is common in S. cerevis- iae and recombination intermediates were found to play an important role for the faithful transmission of mtDNA in this organism [5,6]. Initially identified through mutations that abolish the biased transmission of hypersuppressive mtDNA [7], the CCE1 (MGT1) gene was shown to encode a Holliday junction-resolving enzyme that functions exclu- sively in mitochondria [8]. The loss of this activity in S. cerevisiae cce1 (mgt1) mutants resulted in the accumula- tion of Holliday junctions in mtDNA [5] but the pathway leading to the formation of the intermediates and their role in mtDNA transmission is not fully understood. Homo- logous recombination and replication are two of the most fundamental processes in living cells and are tightly interconnected [9,10]. CCE1 could participate in a recom- bination pathway associated with mtDNA replication, such as intramolecular recombination (recombination with sister chromatid) or in double-strand break repair. It could also be involved in the initiation of recombination-dependent replication events in mtDNA [4,11]. CCE1 has a particular effect on the partitioning of mtDNA and it has been proposed that several mitochondrial genomes linked via recombination junctions constitute the mtDNA heritable unit in S. cerevisiae, whose size is affected directly by CCE1 [5]. However, a recent study of the role of recombination in mtDNA inheritance in S. cerevisiae showed that budding cells were enriched in linear monomers of mtDNA. It was proposed that in addition to a role in initiating a rolling- circle DNA replication, recombination could be instrumen- tal in resolving concatamers into linear monomers in the process of mtDNA partitioning and transmission into buds [11]. Several groups identified the Ydc2 protein of Schizosac- charomyces pombe as a homologue of CCE1 [12–14]. Like CCE1 [7,15], Ydc2 (also called SpCCE1) localized exclu- sively in mitochondria and did not affect nuclear DNA Correspondence to I. Tsaneva, Department of Biochemistry, University College London, Gower St., London WC1E 6BT, UK. E-mail: tsaneva@biochem.ucl.ac.uk Abbreviations: MtDNA, mitochondrial DNA; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; MTS, mito- chondrial targeting signal. (Received 21 February 2003, revised 28 April 2003, accepted 12 May 2003) Eur. J. Biochem. 270, 2837–2847 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03661.x recombination or repair [16]. Ydc2-deficient cells accumu- latedanaggregatedformofmtDNA,whichcouldbe resolved by treatment with Ydc2 in vitro and therefore contained recombination intermediates [16]. The 19.4-kb linear mtDNA genome of S. pombe is found predominantly as linear concatamers with an average size of 100 kb [17]. The pattern of replication intermediates analysed by two- dimensional gel electrophoresis was consistent with a rolling-circle replication mechanism, similar to T4 phage replication [17]. A signal consistent with Holliday junction intermediates was also observed in this study suggesting replication-associated recombination events [17]. The con- servation of CCE1 between the two yeasts and the existence of homologues in the genomes of other fungal species [18,19] is evidence for a conserved pathway, which may well operate in the mitochondria of other eukaryotes and in plant chloroplasts. CCE1 and Ydc2 belong to a group of enzymes that show high specificity for the structure of the Holliday junctions, together with a limited sequence preference for cleavage [12–14,20–23]. Both CCE1 and Ydc2 bind the junction as dimers and manipulate its global conformation from the stacked folded form which prevails in the presence of divalent cations [24], into an unfolded square-planar configuration [14,20]. Binding of CCE1 results in the complete unpairing of the four base-pairs in the centre of the junctions with a lesser disruption of the next base-pairs [19], which is reminiscent of the structure of the junction bound to RuvA [25]. The crystal structure of Ydc2 solved recently revealed the molecular basis of junction cleavage and threw some light on the mechanism of binding and manipulation of the junction [26]. The core of the enzyme was found to be structurally homologous to the bacterial resolvase RuvC and, like all other resolvases, it formed a well-defined dimer. The cata- lytic site consisted of two aspartates ) Asp46 and Asp230 ) which mapped to the catalytic site of RuvC. Protruding from opposite sides of the dimer were two a-helical bundles (triple-helix domains) that had no equi- valents in RuvC. The helical bundles comprised a-helices 1 and 2 from the N-terminus packed to the C-terminal a-helix 8. The high number of positively charged residues and their spatial arrangement suggested that these bundles might be DNA-binding sites [26]. Based on the analysis of the Ydc2 atomic structure we proposed a model for the interaction of Ydc2 with the junction. In this model two arms are engaged in the catalytic sites for cleavage while the other two arms interact with the helical bundles at the sides of Ydc2 [26]. An interesting aspect of this model is that Ydc2 may be a functional fusion of RuvC and RuvA, predisposing the junction for cleavage and, perhaps, branch migration. The proposed DNA binding role of the triple-helix domain of Ydc2 is further supported by the observation in the NCBI database that the N-terminal region of Ydc2 (residues 1–35) contains a small putative DNA-binding SAP motif (after SAF-A/B, Acinus and PIAS) associated with proteins involved in chromosomal organization and DNA repair [27]. In this study we probed the validity of this model by constructing truncated Ydc2 mutants that lacked the SAP motif (helices 1 and 2) or the entire triple-helix motif and tested their biochemical properties in vitro as well as their effect on the localization of Ydc2 to mitochondria and the state of mtDNA in vivo. The results presented in this study show that the truncated mutants were unable to cleave synthetic junctions and were severely impaired in their ability to bind to the junction in vitro. The truncations did not prevent the localization of Ydc2 to mitochondria but the mutants showed no activity in vivo. Significantly, the loss of Ydc2 led to depletion in mtDNA content, which is the first observation of any physiological effect of Ydc2 in vivo. Materials and methods Plasmids and strains The S. pombe strains used in this study FO101 (h- his3-D1 ura4-D18 leu1-32) and its derivative FO362 (h- ydc2:: ura4+ D1 ura4-D18 leu1-32), were kindly provided by F. Osman (Oxford University, UK). The complete medium was yeast extract medium supplemented with 225 mgÆL )1 uracil, leucine and histidine (YES). The minimal medium was Edinburgh Minimal Medium supplemented with appropri- ate amino acids (EMM). The pMW217 plasmid, carrying the full-length YDC2 cDNA tagged with green fluorescent protein (GFP) [16] was kindly provided by M. Whitby (Oxford University, UK). The pREP41/42-enhanced GFP (EGFP) C-terminal expression vectors [28] were kindly provided by I. Hagan (University of Manchester, UK). Expression vector pET21b(+) was from Novagen. The ydc2D mutant strain JAL01 was constructed by gene replacement using a knockout construct containing about 1kboftheydc2 upstream and downstream regions ligated on either side of the ura4 + gene. The knockout fragment was used to transform strain FO101 and select Ura+clones. The genuine replacement of ydc2 by ura4 sequences in the ydc2 genomic locus was checked and verified by Southern blot analysis and PCR. Ydc2 expression plasmids Constructs for the expression of truncated Ydc2 mutants were engineered by PCR amplification creating a deletion of 35 amino acids from the N-terminus (Ydc2-ND35) and deletions of 35 amino acids from the N-terminus plus 15 amino acids from the C-terminus (Ydc2-ND35/CD15) (Fig. 1). Restrictions sites in the primers included an NdeI restriction site and a start codon at the N-terminus for both constructs. The open reading frames were inserted in pET21b(+) as an NdeI–BamHI fragment for Ydc2-ND35 to give plasmids pBS101, and an NdeI–XhoIfragmentfor Ydc2-ND35/CD15 to give plasmid pBS102. In both con- structs the stop codons were deleted to produce in-frame fusions with the C-terminal His6 tag in the vector. All recombinant constructs were verified by DNA sequencing. Construction of truncated and mutant YDC2–EGFP fusions The pREP41–EGFP C-terminal expression vector [28] was used for the generation of Ydc2–EGFP fusion constructs. The 0.662-kb insert from pBS101 was excised with NdeIand BamHI enzymes and ligated into pREP41–EGFP, to give pBS201 (pREP41-YDC2 ND35–EGFP). The 0.617-kb insert from pBS102 was excised with NdeIandXhoI 2838 B. Sigala and I. R. Tsaneva (Eur. J. Biochem. 270) Ó FEBS 2003 enzymes and ligated into pREP41–EGFP, to give pBS202 (pREP41-YDC2 ND35/CD15–EGFP). Single amino acid changes in the Ydc2 open reading frame in plasmid pMW217 were engineered using the Quick ChangeÒ system (Stratagene), producing plasmids pBS203 with a D46N mutation and plasmid pBS204 containing a D230N mutation. All constructs were verified by sequencing. Fig. 1. Ydc2 and RuvC are structural homologues. (A) Superimposition of the structure of Ydc2 (red) and E. coli RuvC (green). The structures were superimposed using secondary structure alignment program [40]. White arrows indicate schematically the positions of the truncations in Ydc2 outside the common structural core. (B) Alignment of the SAP motif of Ydc2 (1 KCF A) and the SAP consensus sequence, as presented in the NCBI database. (C) Schematic representation of the 6 · His and GFP fusion constructs for expression of wild-type Ydc2 and truncated mutants, showing helices 1, 2 and 8 (red bars) and their corresponding amino acid numbers. The GFP is shown schematically in green. Ó FEBS 2003 Holliday junction resolvase Ydc2 in mtDNA maintenance (Eur. J. Biochem. 270) 2839 Expression and purification of truncated Ydc2 recombinant proteins The truncated Ydc2 proteins were expressed in Escherichia coli strain BL21(DE3)Gold. A significant amount of the recombinant protein was insoluble but solubility was greatly improved by inducing the cells at 30 °C and using 0.2% C12E8 (polyoxyethelene 8 lauryl ether) detergent, as described below. One litre of cells containing the expression plasmid were grown in Luria–Bertani medium containing ampicillin (100 lgÆmL )1 )at37°CtoanD 600 of 0.5. Isopropyl b- D -thiogalactoside was added to a final concen- tration of 1 m M and the cells were incubated for an additional 4 h at 30 °C. The cells were collected by centrifugation at 5000 g for 15 min and the pellets were resuspended in 20 mL buffer A (50 m M sodium phosphate pH 8.0, 1 M NaCl). Cells were lysed by sonication (3 · 60 s bursts on ice) and lysozyme was added at 0.75 mgÆmL )1 final concentration. The extracts were incubated on ice for 10 min, followed by addition of 0.2% C12E8 detergent and incubated for further 10 min on ice. The extracts were clarified by centrifugation at 40 000 g, before loading onto Talon columns (Clontech) equilibrated with buffer A. The purification protocol was the same for both truncated Ydc2 proteins. The column was washed with buffer B (buffer A with 20 m M imidazole pH 7.5), before eluting with buffer C (buffer A with 500 m M imidazole pH 7.5). Talon fractions containing the truncated Ydc2 proteins were pooled and dialysed against buffer D [50 m M Tris/HCl pH 7.5, 0.1 M NaCl, 5 m M EDTA, 1 m M dithiothreitol and 10% glycerol]. Protein yields were  1 mg protein from 2 L induced cells. For binding experiments the proteins were further purified by chromatography on SP Sepharose column in buffer D. The fractions were analysed by SDS/PAGE on 12% acrylamide gels stained with Coomassie brilliant blue and the truncated Ydc2 proteins were essentially pure by visual inspection. Junction cleavage assays The junction resolvase activity was assayed by using the four-way junction X12 labelled with 32 Patthe5¢-end of strand 1, as described previously [12]. One ng junction DNA was incubated with the indicated amounts of wild-type or truncated Ydc2 proteins, in cleavage buffer (50 m M Tris/ HCl pH 8.0, 15 m M MgCl 2 ,0.5mgÆmL )1 BSA, 1 m M dithiothreitol). Following incubation at 30 °C for 30 min, the reactions were terminated by adding 5· stop mix (2.5% SDS, 200 m M EDTA, 10 mgÆmL )1 proteinase K) and incubated for further 10 min at 37 °C. Products were analysed by electrophoresis on 10% polyacrylamide dena- turing gels containing 7 M urea, and visualized using a Fujifilm FLA-2000 Phosphorimager. Binding assays Reaction mixtures (10 lL) contained 0.5 ng 32 P-labelled four-way junction in binding buffer (50 m M Tris/HCl pH 8.0, 1 m M dithiothreitol, 200 m M NaCl, 0.1 mgÆmL )1 BSA, 6% glycerol) containing 5 m M EDTA or 5 m M MgCl 2 as described by Oram et al. [12]. After the addition of protein the reactions were incubated for 15 min on ice and loaded onto a 4% native polyacrylamide gel in low ionic buffer (6.7 m M Tris/HCl pH 8.0, 3.3 m M sodium acetate, and 2 m M EDTA or 200 l M MgCl 2 as indicated). Electrophoresis was typically for 1 h and 30 min at 10 VÆcm )1 . For all experiments gels and buffers were precooled at 4 °C and the electrophoresis was carried out at 4 °C. Gels were dried and visualized using a Fujifilm FLA-2000 phosphoimager. Immunofluorescence microscopy S. pombe strain JAL01 was transformed by the lithium chloride method [29] with pREP41–EGFP [28], pMW217 [16], pBS201, pBS202, pBS203 or pBS204. Leu + trans- formants were selected on EMM lacking leucine in the presence of thiamine for inhibition of the nmt promoter. For visualization of Ydc2–GFP constructs, yeast cultures were grown in selective media in the absence of thiamine for 18 h at 30 °C, as described [16]. Cells were harvested by centrifugation at 5000 g, resuspended in water and exam- ined under a fluorescence microscope. For additional staining with the mitochondrion-specific dye MitoTracker Red CMXRos (Molecular Probes), harvested cells were washed once with water, resuspended in 100 n M Mito- Tracker and incubated at room temperature for 15 min. The cells were washed three times with water and examined under a fluorescence microscope (Axioplan 2, ZEISS). Agarose gels electrophoresis of mitochondrial DNA Total cellular DNA was isolated according to Beach and Klar [30]. Following treatment with XhoI, reaction mixtures (20 lg) were loaded onto 0.6% agarose gels in 1 · TAE buffer and electrophoresed at 10 VÆcm )1 for 16 h. The gels werestainedwithethidiumbromideandwerethenblotted onto nylon membranes (BioRad). A part of the ATPase 6 gene from the S. pombe mitochondrial genome was used as mtDNA-specific hybridization probe as described [16] and was kindly provided by M. Whitby (Oxford University, UK). The ura4 + gene was excised from the pREP42–EGFP vector and was used as nuclear DNA hybridization probe. Hybridization probes were labelled with [a- 32 P]dCTP by random priming. A quantitative Southern hybridization analysis of mtDNA was performed by dot blotting. For each strain three dilutions of total cellular DNA ranging between 0.5 lgand10 lg were blotted on nylon membranes (BioRad). The dot blots were hybridized with ATPase 6 gene probe first and the mtDNA hybridization signal was measured using a Fujifilm FLA-2000 phosphorimager. The blots were stripped, re-probed with the ura4 + probe and the hybridization quantified as above. The relative mtDNA content was calculated as described in [11], i.e. the mtDNA hybridization signals were normalized by the signals obtained with the nuclear probe. Results Biochemical activity of truncated Ydc2 mutants Comparison of the refined structure of Ydc2 with the CATH structural database identified E. coli RuvC structure as the most significant match [26]. Superimposition of the 2840 B. Sigala and I. R. Tsaneva (Eur. J. Biochem. 270) Ó FEBS 2003 two structures is shown in Fig. 1. One major difference between RuvC and Ydc2 lies in the small triple-helix motif formed by N-terminal helices 1 and 2 and the last third of the C-terminal helix 8. Two helical bundles protrude on opposite sides of the S-shaped Ydc2 dimer and present positively charged residues on the surface that could bind DNA and could provide a platform for the unstacked square-planar conformation of the Holliday junction [26]. The putative DNA-binding SAP motif encompasses a-helices 1 and 2 at the N-terminus (residues 1–35), as shown schematically on Fig. 1. The two helices form intimate interactions with the C-terminus of a-helix 8. To investigate the functional role of the triple-helix domain, we engineered expression constructs for Ydc2 mutants deleting either the SAP motif alone (residues 1–35) or both the SAP motif and part of a-helix 8 (residues 244– 258), as shown schematically in Fig. 1. The recombinant proteins containing a C-terminal 6 · His affinity tag were expressed in E. coli and purified to near homogeneity. The CD spectra of the truncated mutants exhibited the charac- teristic pattern of a-helical secondary structure indicating predominantly folded structure. The CD spectra of Ydc2 and the Ydc2-ND35/CD15 double truncation mutant were nearly identical (data not shown). The ability of the truncated Ydc2 mutants to resolve Holliday junctions in vitro was tested using the synthetic four-way junction X12 [12] and the formation of cleavage products was examined by both native and denaturing PAGE. Neither Ydc2-ND35, lacking the first 35 residues from its N-terminus, nor Ydc2-ND35/CD15, carrying a second truncation of 15 residues at the C-terminus, were able to resolve the synthetic junction (Fig. 2 and data not shown). As shown in Fig. 2 for strand one, the denaturing PAGE showed no evidence for uncoordinated cleavage of this strand, which is one of the preferred strands for resolution. No cleavage activity could be detected using another synthetic junction (Jbm5 [20], data not shown). Binding of truncated mutants to Holliday junctions The complete loss of resolvase activity of the truncated mutants, which retained all amino acid residues that map to the structural core shared with RuvC including the catalytic site, could be due to a defect in binding to the Holliday junction. To investigate the interactions of the truncated Ydc2 proteins with Holliday junctions, we carried out electrophoretic mobility shift experiments with junction X12. Control Ydc2 formed two protein–DNA complexes (Fig. 3A, lane b) as shown previously [12,13,23]. Complex I contains one dimer of Ydc2 while complex II most likely results from the binding of two dimers of Ydc2 and its functional significance is doubtful [14]. The Ydc2-ND35 mutant showed a severely impaired binding to the junction (Fig. 3A, lanes c–g), which appeared mostly as a smear of high molecular weight species. The formation of complex I was not well defined and could only be seen at high protein concentrations (Fig. 3A, lane g). A high molecular weight complex could be observed at concentrations of the mutant protein above 50 n M (Fig. 3A, lanes d–f) but not with wild-type Ydc2. The composition and architecture of this complex cannot be determined in these assays but it is unlikely that it represents interactions with the junction that are functionally relevant. These results indicate that deletion of the SAP motif alone affected greatly the ability of the truncated protein to bind the junction correctly. The binding patterns of smeared high molecular weight com- plexes also suggested unstable and abnormal interactions. The binding of the double mutant Ydc2-ND35/CD15 to Holliday junctions was better defined: complex I formed more readily and complex II could be detected at higher protein concentrations (Fig. 3B, lanes d–h) with less high molecular weight smear (Fig. 3B). However, the junction- binding affinity of Ydc2-ND35/CD15 appeared to be at least an order of magnitude lower than that of wild-type Ydc2 (compare lanes b and g, and c and h in Fig. 3B). No binding to liner duplex DNA was observed using either of the truncated proteins under any of the conditions tested (data not shown). The improved binding pattern of the double mutant, compared to Ydc2-ND35 suggested that removal of the N-terminal helices 1 and 2 while leaving the interacting a-helix 8 intact, may destabilize the proteins leading to spurious protein–protein or/and protein–DNA interactions. The crystal structure of Ydc2 shows that deletion of the two N-terminal helices 1 and 2 would expose to the solvent a significant part of the C-terminal a-helix 8 which could destabilize the protein in solution. Consistent with this, the solubility of the Ydc2-ND35/CD15 mutant protein in E. coli was significantly better than that of the Ydc2-ND35 mutant. Ydc2 binding imposes a square-planar configuration on the Holliday junction counteracting the junction-folding effect of divalent cations. If binding by the triple-helix motif Fig. 2. Truncated Ydc2 mutants do not show resolvase activity in vi tro. Denaturing PAGE showing reactions with junction X12 32 P-labelled on strand 1 [11] incubated with increasing amounts of Ydc2-ND35 (left panel) or Ydc2-ND35/CD15 (right panel). Lanes d–f contained 0.57, 0.87 and 1 l M Ydc2-ND35, lanes g–h contained 0.6, 1.2, 3 and 4 l M Ydc2-ND35/CD15, respectively. Controls contained no protein (X12), 0.6 l M purified Ydc2-His6 (Ydc2) or 2 lLCce1-GSTfusionprotein (Cce1)expressedinyeastcellsandpurifiedasdescribedin[41]. Ó FEBS 2003 Holliday junction resolvase Ydc2 in mtDNA maintenance (Eur. J. Biochem. 270) 2841 were important for manipulating the global conformation of the junction, then the deletion of this motif would have an even more serious effect on Ydc2 binding in the presence of divalent cations. We tested the effect of Mg 2+ on the binding of the double mutant Ydc2-ND35/CD15 to junction DNA, as this mutant showed a well-defined complex I in the absence of divalent cations. In the presence of Mg 2+ complex I and complex II were readily observed with the wild-type protein, along with junction cleavage products (Fig. 3C, lanes b and c). Under these conditions the binding of the double mutant was severely reduced. Traces of complex I could be detected at concentrations above 100 n M Ydc2-ND35/CD15 (lanes f and g) but although some binding to the junction could occur in the presence of Mg 2+ it was either not sufficient or not functional for resolution. If binding of two arms of the junction to the triple-helix motif was required to hold the junction in a square-planar conformation, it was interesting to test whether RuvA binding to the junction could substitute for the function of the protruding bundles, as it was shown previously that wild-type Ydc2 readily binds to and cleaves junction that is already bound by a RuvA tetramer [22]. Pre-binding of RuvA had no effect on the ability of Ydc2-ND35/CD15 to bind the junction, nor did the presence of RuvA lead to cleavage of the junction (data not shown). These observa- tions suggest that interactions between the Holliday junc- tion and the helical bundles of Ydc2 may be required for the cleavage step to occur. The SAP motif most likely participates in junction binding, as deletion of this motif affected profoundly the activity of the protein. However, interactions with helix 8 could play an important functional role, such as the correct positioning of the SAP motifs in relation to the rest of the protein as well as for the stability of the protein. The truncated Ydc2 mutant proteins localize to the mitochondria in S. pombe Ydc2 (30.2 kDa) fused to the ORF of GFP was shown to localize in the mitochondria of S. pombe [16]. Scanning the full-length Ydc2 amino acid sequence with the PSORT II program [31] gave a weak prediction for a possible mitochondrial targeting signal (MTS) on the N-terminus of the protein and a predicted cleavage at amino acid 33. A possible MTS in this part of the protein would be incompatible with the proposed DNA-binding role. To investigate the possible role of the a-helical bundle in mitochondrial targeting, we constructed yeast expression plasmids for Ydc2-ND35 or Ydc2-ND35/CD15 fusions with EGFP using plasmid pREP41–EGFP [28]. This vector gives a medium strength expression in S. pombe under the control of the nmt promoter. Representative images of yeast cells expressing the fusion proteins are shown in Fig. 4. It is clear that both Ydc2(ND35)–EGFP and Ydc2(ND35/CD15)– EGFP fusion proteins colocalize with the mitochondria (right panel). These results show that no mitochondrial targeting sequence exists in the portions deleted from the N or C termini of the protein. There is probably an internal targeting signal, which has not been identified at present. Effect of Ydc2 mutants on mtDNA in vivo The correct localization of the truncated Ydc2–GFP fusion proteins to yeast mitochondria made it possible to investi- gate their effect on yeast mtDNA in vivo. The truncated mutants were tested alongside Ydc2–GFP fusions carrying point mutations in the catalytic site (D46N or D230N), which abolish the cleavage activity of the enzyme but do not affect the specific binding to the junction [26]. For these experiments we used ydc2D S. pombe strain JAL01, con- structed as described in Materials and methods. Similar to the insertion-inactivated ydc2 mutant FO362 [16], ydc2D strain JAL01 showed no discernable DNA repair or growth phenotype (Judit Arenas-Licea, unpublished data). Total Fig. 3. Binding of truncated Ydc2 mutants to 32 P-labelledjunctionX12. Electrophoretic mobility binding assays with increasing concentrations of control Ydc2 and Ydc2-ND35 (A) or Ydc2-ND35/CD15 (B), as indicated, visualized by phosphorimaging. (C) Binding reactions with increasing concentrations Ydc2-ND35/CD15inthepresenceof5m M MgCl 2 , as described in Materials and methods. 2842 B. Sigala and I. R. Tsaneva (Eur. J. Biochem. 270) Ó FEBS 2003 DNA prepared from both strains showed a significant increase in the amount of DNA retained in the wells of ethidium bromide-stained agarose gel (Fig. 5A), as observed in Doe et al. [16]. This material could not be released by treatment with XhoI, which cleaves S. pombe mtDNA at a single site to produce linear monomers. Southern blot hybridization with mtDNA-specific probe (part of the ATPase 6 gene), revealed a 19.4-kb band in the XhoI-treated DNA from wild-type FO101 cells (Fig. 5B, lane e). This band, which corresponds to the monomer size of linear mtDNA, was greatly reduced in the DNA from FO362 or JAL01 cells, while the presence of aggregated mtDNA in the wells was clearly observed (Fig. 5B, lanes f and g). In order to test the effect of mutations on the mtDNA ÔphenotypeÕ, expression plasmids carrying wild-type or mutants of Ydc2–GFP were introduced in strain JAL01. Vector pREP41–EGFP in JAL01 was used as control. Expression of the fusion proteins was induced by growth in the absence of thiamine and was verified by fluorescence microscopy. Southern blots of total cellular DNA probed Fig. 4. Subcellular localization of Ydc2 and truncated mutants to yeast mitochondria. Fluorescence micrographs of Ydc2–EGFP fusions in ydc2D S. pombe cells (green), MitoTracker staining of mitochondria (red) and the merged images (yellow). Fig. 5. The state of mtDNA in Sc. pombe is affectedbythelossofYdc2.Agarose gel electrophoresis of undigested or XhoI-digested total cellular DNA from strain FO101 (ydc2 + ), FO362 (ydc2::ura4 + )andJALO1 (ydc2D::ura4 + ) stained with ethidium bromide (A) or blotted and hybridized to mtDNA- specific probe and visualized by phosphor- imaging (B). Ó FEBS 2003 Holliday junction resolvase Ydc2 in mtDNA maintenance (Eur. J. Biochem. 270) 2843 with the ATPase 6 gene is shown in Fig. 6. The undigested mtDNA from control ydc2D cells was detected as a broad smear with some aggregated material in the well (Fig. 6A, lane a). Expression of wild-type Ydc2–GFP (Fig. 6A, lane b) showed a tighter mtDNA size distribution, reduced the amount of aggregated material in the well and gave a significantly stronger hybridization signal, despite equal amounts of total DNA being loaded, as shown by ethidium bromide staining (Fig. 6B). Upon digestion with XhoIa weak 19.4-kb band of linear monomer mtDNA could be detected in the ydc2D DNA sample but a significant amount of mtDNA remained smeared or trapped in the well (lane i). In contrast, XhoI digestion of DNA from cells expressing wild-type Ydc2–GFP resulted in the appearance of a prominent 19.4-kb band and no mtDNA was retained in the well (lane j). Expression of Ydc2–GFP therefore reversed the mtDNA mutant ÔphenotypeÕ of JAL01 cells. This result clearly showed that the fusion Ydc2–GFP was active in vivo and the fusion constructs could therefore be used to test the mutants. Expression of the single or double truncated mutants Ydc2(ND35)–GFP and Ydc2(ND35/CD15)–GFP in JAL01 had little effect on the mtDNA profile, which was essentially the same as ydc2D cells carrying the vector control, both before and after XhoI digestion (Fig. 6). Increasing amounts of total cellular DNA were loaded on the gels, as the hybridization signal in these samples was very low. The general pattern remained the same: the 19.4-kb band was weak and a significant amount of mtDNA remained trapped in the wells. These results showed no evidence for any residual biological activity of the truncated mutants in vivo. In addition, we also expressed in ydc2D cells catalytically inactive point mutants of Ydc2, namely D46N and D230N [26]. The Southern blots consistently showed some differences in the mtDNA profile of the point mutants compared to the truncated mutants and the vector control. The 19.4-kb band was nearly as prominent as seen with wild-type Ydc2 (Fig. 6C). However, the mtDNA hybrid- ization signal was weaker compared to cells expressing wild-type Ydc2. As shown in the experiments below, Ydc2- deficient cells suffered an overall depletion of mtDNA content and this defect could not be complemented by either the point mutants or the truncated mutants. The observa- tion in the Southern blots of more unit length mtDNA released by XhoI digestion suggests that binding of the Ydc2 point mutants to Holliday junctions affects subsequent steps in the processing of unresolved recombination intermedi- ates. It seems likely that these intermediates are subject to fragmentation and degradation in the cells. Binding of the catalytically inactive mutants to Holliday junctions may prevent such degradation. The truncated mutants, on the other hand, had no such effect, which may be an indication for impaired binding to Holliday junctions in vivo. MtDNA depletion in Ydc2-deficient yeast cells The Southern analysis suggested that the content of mtDNA was reduced in Ydc2-deficient cells. To investigate this effect further the mtDNA content was measured by quantitative dot blot hybridization of total DNA, as described in Materials and methods. The relative amount of mtDNA was expressed as the ratio between mtDNA- specific and nuclear hybridization signals. As shown in Fig. 7A, the relative amount of mtDNA in Ydc2-deficient cells was about three to four times lower than in the wild- type control. The lack of Ydc2 therefore caused a significant depletion of mtDNA in S. pombe. This result did not depend on the growth conditions, as similar levels of mtDNA depletion were observed with cells grown in rich media (data not shown). Expression of wild-type Fig. 6. MtDNA profile of Ydc2 mutants. (A) Southern blot hybridization of undigested (lanes a–h) or XhoI-digested DNA (lanes i–p) isolated from induced ydc2D cells JAL01 carrying plasmid pREP41–GFP (vector), plasmids pMW207 expressing wild-type Ydc2– GFP (ydc2 + ), pBS201 (N35D mutation) or pBS202 (N35D /C15D mutation). (B) Total DNAin(A)stainedwithethidiumbromide. Lanes contained 20 lg(1·), 40 lg(2·)or 60 lg(3·)ofDNA.(C)XhoI-digested DNA from induced JAL01 cells carrying pREP41– GFP (vector), wild-type Ydc2–GFP (ydc2 + ), pBS203 (D46N mutation), pBS204 (D230N mutation) and pBS202 (N35D/C15D mutation). Hybridization was with 32 P- labelled mtDNA probe (from the ATPase 6 gene) and the blot was visualized with a phosphorimager. 2844 B. Sigala and I. R. Tsaneva (Eur. J. Biochem. 270) Ó FEBS 2003 Ydc2–GFP restored the mtDNA content of the ydc2D cells to nearly wild-type levels but the truncated mutants did not (Fig. 7A). Similarly, the catalytically inactive mutants D46N and D230N could not complement the mtDNA deficiency of ydc2D cells. These results show clearly that maintaining a normal content of mtDNA in fission yeast depended on the catalytic activity of Ydc2. The effect of expressing wild-type or mutant Ydc2–GFP in ydc2 + yeast cells was also examined in order to test for dominant-negative effects of truncated and point mutants. The results of this experiment are shown in Fig. 7B. Expression of wild-type Ydc2–GFP resulted in a clear reduction of the mtDNA content of the cells. Similar decrease in the mtDNA content was observed in cells expressing the catalytically inactive point mutants and the truncated mutant Ydc2(ND35/CD15)–GFP. These results therefore indicate both a gene dosage effect exerted by the wild-type Ydc2 and dominant-negative effects exerted by the mutants. The detrimental effect of Ydc2 over-expression on mtDNA maintenance is consistent with the observed deleterious effects of over-expressing a bacterial resolvase in yeast nuclei [32] and may be due to nonspecific nuclease activity or cleavage of replication intermediates. The dominant-negative effect of the mutants was not unex- pected. The catalytically inactive point mutants, in parti- cular, retain their full ability to bind Holliday junction and would therefore interfere with resolution. The effect of the truncation mutant appeared to be slightly smaller. This observation correlates with its defect in junction binding – it would be a poor competitor for wild-type Ydc2. The magnitude of the dominant-negative effects is difficult to assess, however, without measuring the expression levels of the mutants relative to the endogenous Ydc2 and the effect of GFP on the specific activity of the fusion proteins. All three mutants could also form mixed dimers with the endogenous protein which would produce a dominant- negative effect. Theresultsofthealltheexperimentsin vivo clearly showed that truncated Ydc2 mutants lacking the SAP motif alone or the whole triple-helix domain were not functional in yeast mitochondria. Discussion In this paper we investigated the role of the triple-helix domain for Ydc2 function using truncated mutants designed to retain the amino acid residues that constitute the structural core shared with RuvC. The immunofluores- cent microscopy observations clearly demonstrated that the truncated Ydc2 mutants fused to GFP localized exclusively in the mitochondria (Fig. 4), which ruled out any role of the helical bundle in mitochondrial targeting. The two truncated mutants, Ydc2-ND35 and Ydc2- ND35/CD15, showed no cleavage activity on Holliday junctions in vitro. The loss of activity is most likely due to the mutants’ defects in binding to junction DNA. Ydc2- ND35/CD15 in particular showed a binding pattern similar to wild-type Ydc2 but the affinity for the junction was significantly reduced (Fig. 3B). As discussed below, the truncated mutants showed no evidence for activity in vivo. Our results are therefore fully consistent with the proposed DNA binding role for the triple-helix domain. Moreover, the results imply that binding to these domains plays a crucial role for junction resolution. While the SAP motif at the N-terminus most likely mediates binding to the junction, its interaction with the C-terminal helix 8 could be important for cleavage to occur. In the model proposed previously interactions of two arms of the junction with the protruding helical bundles of Ydc2 would provide the platform for maintaining the square-planar configuration needed for cleavage. While in Fig. 7. Ydc2 mutations affect mtDNA content in S. pombe. Quantita- tive dot blot hybridization analysis of mtDNA. (A) Relative mtDNA content in induced ydc2D JAL01 cells carrying pREP41–EGFP vector alone (vector), and plasmids pMW217 (wild-type Ydc2–GFP), pBS201 (N35D mutation), pBS202 (N35D/C15D mutation), pBS203 (D46N mutation), and pBS204 (D230N mutation), as indicated. Strain FO101 (ydc2 + ) with Ydc2–GFP vector was used as control. (B) Rel- ative mtDNA in ydc2 + cells. Strain FO101 carrying Ydc2–GFP vec- tor, plasmids pMW217 (wild-type Ydc2–GFP), pBS202 (N35D/C15D mutation), pBS203 (D46N mutation), and pBS204 (D230N mutation), as indicated. The relative amount of mtDNA was expressed as the ratio between mtDNA and nuclear hybridization signals, as described in Materials and methods. Ó FEBS 2003 Holliday junction resolvase Ydc2 in mtDNA maintenance (Eur. J. Biochem. 270) 2845 the RuvABC system this configuration would be imposed by RuvA, the additional DNA-binding domain would equip Ydc2 with a combined RuvA–RuvC function. The presence of RuvA had no effect on the binding or cleavage activity of the truncated Ydc2 mutants in vitro (data not shown). It may be that precise interactions of the triple-helix domain with the arms of the junction are needed for the correct positioning of the catalytic sites for cleavage, which may involve the steric ÔpinÕ ) the loop protruding between helices 3 and 4 at the junction crossover – and the unpairing of four base-pairs in the centre of the junctions [19]. Detailed understanding of the functional role of these interactions would require the crystal structure of the Ydc2–junction complex. In this study the function of several Ydc2 mutants, expressed as fusions with GFP, was examined in vivo. An important result from these experiments was the finding that loss of catalytically active Ydc2 caused a significant depletion of mtDNA in S. pombe (Fig. 7). Our results are consistent with the observation of a reduced number of chondriolites in Ydc2-deficient cells [16]. Although the disruption of Ydc2 did not present a discernible phenotype in this petite-negative organism [16] the maintenance of mtDNA was clearly disturbed by the absence of Ydc2. Similar experiments in S. cerevisiae did not find a signifi- cant effect of cce1 mutants on mtDNA content. However, the double loss of Cce1p and Mhr1p, another protein involved in mtDNA recombination, resulted in the com- plete inability of S. cerevisiae to maintain mtDNA [11]. There is no identifiable Mhr1 homologue in Sc. pombe and it is likely that differences in the pathways of mtDNA maintenance have evolved between the two highly diver- gent yeast species. The role of Ydc2 for maintaining the content of mtDNA in fission yeast is not clear. Apart from DNA repair, recombination could be involved in the initiation of mtDNA replication (recombination-dependent replication [9,33]) by a rolling-circle mechanism coupled to concatamer resolution [4,11,17]. In this case the loss of Ydc2 would directly affect the number of mtDNA molecules replicated and hence the number of molecules that are inherited. If mtDNA transmission involved converting concatamers into monomers, as suggested for S. cerevisiae [11], Ydc2 could also play a role in a parsing pathway that ensures and controls the partitioning of mtDNA in daughter cells. Recombination functions, including Holliday junction- processing enzymes, are likely to affect the metabolism of mtDNA in other eukaryotes including higher eukaryotes. There is substantial evidence that mtDNA in plants replicates via a rolling-circle mechanism involving Holliday junctions [34–36]. The malaria parasite Plasmodium falci- parum mtDNA undergoes recombination in conjunction with replication and most likely replicates via a mechanism that is largely dependent on recombination of linear tandem arrays, generated by a rolling-circle process [4,37]. Interest- ingly, the formation of Holliday junctions in mtDNA from human heart muscle was recently demonstrated [38] suggesting that recombination intermediates and enzymes that resolve them could be involved in maintaining the stability and inheritance of mitochondrial genomes in human cells. These may have important implications for mitochondrial dysfunction and mutations in mtDNA associated with mitochondrial diseases and implicated in aging (reviewed in [39]). Understanding the molecular mechanisms and pathways of mtDNA inheritance in fission yeast, alongside budding yeast and other eukaryotic species, could help to elucidate some general mechanisms conserved in the evolution. Acknowledgements We would like to thank F. Osman (presently in the University of Oxford, UK) for yeast strains, plasmids and expertise in the course of these experiments, A. Pittman for help with the cloning of the mutants, M. Whitby (University of Oxford, UK) and I. Hagan (University of Manchester, UK) for providing plasmids. Strain JAL01 was construc- ted by J. Licea-Arenas and M. Oram. We thank T. Barrett and L. Pearl from the Institute of Cancer Research, Chester Beatty Laboratories, for their advice on engineering Ydc2 truncations and T. Barratt for contributing to Fig. 1. We gratefully acknowledge the financial support of the Wellcome Trust (Ref. no. 041244). References 1. McGlynn, P. & Lloyd, R.G. (2002) Genome stability and the processing of damaged replication forks by RecG. Trends Genet. 18, 413–419. 2. Robu, M.E., Inman, R.B. & Cox, M.M. (2001) RecA protein promotes the regression of stalled replication forks in vitro. Proc. Natl Acad. Sci. USA 98, 8211–8218. 3. Postow, L., Ullsperger, C., Keller, R.W., Bustamante, C., Volo- godskii, A.V. & Cozzarelli, N.R. (2001) Positive torsional strain causes the formation of a four-way junction at replication forks. J. Biol. Chem. 276, 2790–2796. 4. Williamson, D. (2002) The curious history of yeast mitochondrial DNA. Nature Rev. Genet. 3, 475–481. 5. Lockshon,D.,Zweifel,S.G.,Freeman-Cook,L.L.,Lorimer,H.E., Brewer, B.J. & Fangman, W.L. (1995) A role for recombination junctions in the segregation of mitochondrial DNA in yeast. Cell. 81, 947–955. 6. Piskur, J. (1997) The transmission disadvantage of yeast mito- chondrial intergenic mutants is eliminated in the mgt1 (cce1) background. J. Bacteriol. 179, 5614–5617. 7. Zweifel, S.G. & Fangman, W.L. (1991) A nuclear mutation reversing a biased transmission of yeast mitochondrial DNA. Genetics 128, 241–249. 8. Kleff, S., Kemper, B. & Sternglanz, R. (1992) Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease. EMBO J. 11, 699–704. 9. Kogoma, T. (1997) Stable DNA replication: Interplay between DNA replication, homologous recombination, and transcription, Microbiol. Mol. Biol. Rev. 61, 212. 10. Cox, M.M., Goodman, M.F., Kreuzer, K.N., Sherratt, D.J., Sandler, S.J. & Marians, K.J. (2000) The importance of repairing stalled replication forks. Nature 404, 37–41. 11. Ling, F. & Shibata, T. (2002) Recombination-dependent mtDNA partitioning: in vivo role of Mhr1p to promote pairing of homo- logous DNA. EMBO J. 21, 4730–4740. 12. Oram, M., Keeley, A. & Tsaneva, I. (1998) Holliday junction resolvase in Schizosaccharomyces pombe has identical endo- nuclease activity to the CCE1 homologue YDC2. Nucl. Acids Res. 26, 594–601. 13. Whitby, M.C. & Dixon, J. (1997) A new Holliday junction resolving enzyme from Schizosaccharomyces pombe that is homologous to CCE1 from Saccharomyces cerevisiae. J. Mol. Biol. 272, 509–522. 2846 B. Sigala and I. R. Tsaneva (Eur. J. Biochem. 270) Ó FEBS 2003 [...]... Substrate specificity of the SpCCE1 Holliday junction resolvase of Schizosaccharomyces pombe J Biol Chem 273, 35063–35073 23 White, M.F & Lilley, D.M (1997) Characterization of a Holliday junction- resolving enzyme from Schizosaccharomyces pombe Mol Cell Biol 17, 6465–6471 24 Duckett, D.R., Murchie, A.I & Lilley, D.M (1990) The role of metal ions in the conformation of the four-way DNA junction EMBO J 9,... central disruption of a four-way junction on binding CCE1 resolving enzyme J Mol Biol 296, 421–433 20 Schofield, M.J., Lilley, D.M.J & White, M.F (1998) Dissection of the sequence specificity of the Holliday junction endonuclease CCE Biochemistry 37, 7733–7740 21 White, M.F & Lilley, D.M (1996) The structure-selectivity and sequence-preference of the junction- resolving enzyme CCE1 of Saccharomyces cerevisiae... Crystal structure of an octameric RuvA -Holliday junction complex Molec Cell 2, 361–372 26 Ceschini, S., Keeley, A., McAlister, M.S., Oram, M., Phelan, J., Pearl, L.H., Tsaneva, I.R & Barrett, T.E (2001) Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2 EMBO J 20, 6601–6611 27 Aravind, L & Koonin, E.V (2000) SAP – a putative DNAbinding motif involved in chromosomal organization... Vectors for the expression of tagged proteins in Schizosaccharomyces pombe Gene 221, 59–68 29 Okazaki, K., Okazaki, N., Kume, K., Jinno, S., Tanaka, K & Okayama, H (1990) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe Nucl Acids Res 18, 6485–6489 30 Beach, D.H & Klar, A.J (1984) Rearrangements of the transposable... Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe Mol Gen Genet 263, 889–897 17 Han, Z & Stachow, C (1994) Analysis of Schizosaccharomyces pombe mitochondrial DNA replication by two dimensional gel electrophoresis Chromosoma 103, 162–170 18 Sharples, G.J (2001) The X philes: structure-specific endonucleases that resolve Holliday junctions Mol Microbiol... I.J & Jacobs, H.T (2001) Prominent mitochondrial DNA recombination intermediates in human heart muscle EMBO Report 2, 1007–1012 39 Wallace, D.C (1992) Diseases of the mitochondrial DNA Ann Rev Biochem 61, 1175–1212 40 Orengo, C.A., Brown, N.P & Taylor, W.R (1992) Fast structure alignment for protein databank searching Proteins 14, 139–167 41 Martzen, M.R., McCraith, S.M., Spinelli, S.L., Torres, F.M.,... Kowalczykowski, S.C (2000) Initiation of genetic recombination and recombination-dependent replication Trends Biochem Sci 25, 156–165 34 Backert, S (2002) R.-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1 EMBO J 21, 3128–3136 35 Backert, S., Dorfel, P., Lurz, R & Borner, T (1996) Rolling-circle replication of mitochondrial DNA in the higher plant... 2003 Holliday junction resolvase Ydc2 in mtDNA maintenance (Eur J Biochem 270) 2847 14 Schofield, M.J., Lilley, D.M.J & White, M.F (1997) Sequence specificity of CCE1 Biochem Soc Trans 25, S646–S646 15 Ezekiel, U.R & Zassenhaus, H.P (1993) Localization of a cruciform cutting endonuclease to yeast mitochondria Mol Gen Genet 240, 414–418 16 Doe, C.L., Osman, F., Dixon, J & Whitby, M.C (2000) The Holliday junction. .. Backert, S & Borner, T (2000) Phage T4-like intermediates of DNA replication and recombination in the mitochondria of the higher plant Chenopodium album (L.) Curr Genet 37, 304–314 37 Preiser, P., Wilson, R., Moore, P., McCready, S., Hajibagheri, M., Blight, K., Strath, M & Williamson, D (1996) Recombination associated with replication of malarial mitochondrial DNA EMBO J 15, 684–693 38 Kajander, O.A.,... Rearrangements of the transposable mating-type cassettes of fission yeast EMBO J 3, 603–610 31 Nakai, K & Kanehisa, M (1992) A knowledge base for predicting protein localization sites in eukaryotic cells Genomics 14, 897–911 32 Doe, C., Dixon, J., Osman, F & Whitby, M.C (2000) Partial suppression of the fission yeast rqh(–) phenotype by expression of a bacterial Holliday junction resolvase EMBO J 19, 2751–2762 . Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance Barbara. resulted in the accumula- tion of Holliday junctions in mtDNA [5] but the pathway leading to the formation of the intermediates and their role in mtDNA transmission