Tài liệu Báo cáo khoa học: Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance pptx

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:
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).
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