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INTRODUCTION |
Homologous DNA recombination is ubiquitous in all organisms. It
promotes genetic diversity, plays an important role in DNA repair and
helps maintain genome integrity. Escherichia coli RecA protein plays a central role in homologous recombination pathways and
catalyzes strand exchange between homologous DNA molecules, leading to
the formation of recombination intermediates (1, 2). In these
intermediates, two homologous duplex DNA molecules are joined by a
four-way DNA junction referred to as a Holliday junction (3). E. coli proteins RuvA and RuvB form a protein complex that promotes
branch migration of Holliday junction and facilitates the extension of
heteroduplex DNA region. E. coli RuvC protein resolves a
Holliday junction by endonucleolytic cleavage (4, 5). Recently, it has
been proposed that the three proteins RuvABC form a protein complex
called a resolvasome that efficiently processes Holliday junctions
(6).
Although Holliday junction-resolving activities have been found in
extracts from a wide variety of organisms, E. coli RuvC is
one of the best characterized resolvases. Genetic analyses indicate
that RuvC resolvase plays a defined role in processing recombination
and repair intermediates (7-11). In vitro studies confirm
that RuvC specifically interacts with and resolves Holliday junctions
by endonucleolytic cleavage (12, 13). Using several model substrates,
including synthetic Holliday junctions, the following has been shown.
(i) RuvC is active as a stable dimer that binds a Holliday junction in
the presence or absence of divalent metal ions (13-15). (ii) RuvC
forms a complex with Holliday junctions in which the two continuous
(noncrossover) DNA strands at the junction are hypersensitive to attack
by hydroxyl radicals and permanganate; this indicates a distortion in
the junction DNA and disruption of base pairing at the crossover. This
is likely to be an important characteristic of the interaction between
RuvC and its DNA substrate (14, 16). (iii) In the presence of metal ions such as Mg2+ and Mn2+, RuvC resolves the
junctions by introducing symmetrical nicks in the continuous
(noncrossover) strands (14, 15, 17), and (iv) cleavage occurs
preferentially at the consensus sequence 5'-(A/T)TT
(G/C), where
represents a cleavage site close to the crossover point (18, 19).
Following cleavage, the resolution process is completed by DNA ligase,
which rejoins the 5'-P and 3'-OH termini in the nicked duplex products
(13, 14).
Structural analysis by x-ray crystallography at 2.5 Å resolution
combined with mutation studies also provided further insight into the
reaction mechanism of RuvC-mediated Holliday junction resolution
(20-23). The two subunits in the RuvC dimer are related by a dyad
axis, and each subunit possesses a large cleft, which could accommodate
double-stranded DNA at the Holliday junction (20). The catalytic center
of RuvC includes the four acidic residues Asp-7, Glu-66, Asp-138, and
Asp-141 located at the bottom of the cleft (20, 21). It was recently
shown that the basic residues Lys-107 and Lys-118, located on the wall
of the cleft near the catalytic center, are involved in DNA binding and
play an important role in stabilizing the reaction intermediate during endonucleolytic cleavage by RuvC (22).
Phe-69 lies in the protruding loop preceding the second
-helix and
faces the catalytic center of RuvC (20). A previously isolated F69L
mutant of RuvC showed no detectable cleavage activity, but formed a
dimer that bound a Holliday junction in the same manner as wild-type
RuvC, as judged by a gel shift assay in which no salt was added to the
reaction mixtures (23). This result suggests that Phe-69 does not
directly participate in DNA binding activity detectable by a salt-free
gel shift assay, but that it must play an important role in RuvC
function. We speculated that this residue makes a stacking interaction
with a nucleotide base near the cleavage site (20, 23).
This study examines the role of Phe-69 of RuvC in greater detail. A
series of mutants of Phe-69 were constructed and analyzed in
vivo and in vitro. The results provide direct evidence
that Phe-69 interacts with DNA and contributes to disruption of base paring in the RuvC·DNA complex. This function is critically important for formation of a catalytically competent complex between RuvC and the
Holliday junction. A model is proposed for RuvC-mediated endonucleolytic cleavage and resolution of Holliday junctions.
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EXPERIMENTAL PROCEDURES |
E. coli Strains and Plasmids--
E. coli strain
HRS1200 (
ruvC200::Kmr) is a
derivative of AB1157, a ruv+ control strain
(21). HRS777 is a
ruvC200::Kmr
derivative of BL21 (DE3) and was used for overproduction of mutant RuvC
proteins (21). Plasmid pRC100 (21) carries the
ruvC+ gene in pET-8c, a T7 expression plasmid
(24). The mutant ruvC genes were constructed based on the
method of Kunkel (21) using a site-directed mutagenesis kit (Takara
Shuzo, Kyoto). The codon for Phe-69 (TTT) was altered to that of Tyr
(TAC), Trp (TGG), and Ala (GCT). ruvC gene mutations were
confirmed by sequencing. The F69L mutant had been isolated in a
previous study (23). Each mutant ruvC gene was carried in
pET-8c with the same construction as pRC100. Bacteria were routinely
cultured in Luria-Bertani medium at 37 °C. If necessary,
ampicillin was added to the medium at the final concentration of 50 µg/ml.
UV Light Sensitivity Test--
Exponentially growing HRS1200
(
ruvC) cells harboring ruvC-expression
plasmids, which were suspended in M9 buffer (~2 × 108 cells per ml), were irradiated with various doses of
UV. Cells were plated on Luria-Bertani plates containing ampicillin (50 µg/ml), and the surviving colonies were scored after incubation for
15 h at 37 °C in the dark.
Purification of Wild-type and Mutant RuvC Proteins--
Phe-69
mutant proteins were purified by the same procedure as wild-type and
other mutant proteins, which was described in our previous report (22).
Synthetic Holliday Junctions--
Synthetic Holliday junctions
were prepared by annealing four synthetic oligonucleotides as described
(21). The sequences of synthetic Holliday junctions, X12 and
HJ2, were described elsewhere (22). The
synthetic Holliday junction HJ2L consists of four oligonucleotides,
which are ST-1
(5'-GACGCTGCCGAATTCTACCAGTGCCTGGCTTTGACCTCTTTGCCCACCTGCAGGTTCACCC-3'), ST-2
(5'-TGGGTGAACCTGCAGGTGGGCAAAGAGGTCAATCGGTTGTAAACGTCAAGCTTTATGCCGTT-3'), ST-3
(5'-GAACGGCATAAAGCTTGACGATTACAACCGATTCTGGAGCTGTCTAGAGGATCCGACTATCGA-3'), and ST-4
(5'-ATCGATAGTCGGATCCTCTAGACAGCTCCAGAAAGCCAGGCACTGGTAGAATTCGGCAGCGT-3'). The sequences underlined are the same junction core sequences as
those of HJ2. HJ2L was used as a substrate for a potassium permanganate
footprinting experiment.
Cleavage and Gel Shift Assays--
Holliday junction cleavage
and gel shift assays were performed essentially as described previously
(21). In brief, reaction mixtures (20 µl) contained 10 ng of a
32P-labeled synthetic Holliday junction, 20 mM
Tris acetate, pH 8.0, 10 mM (for cleavage assay) or 0.5 mM (for gel shift assay) magnesium acetate, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 5%
(v/v) glycerol, and the indicated concentrations of RuvC. For a
standard cleavage assay, the mixtures were incubated at 37 °C for 30 min, and then the samples were analyzed by 9% native polyacrylamide
gel electrophoresis (PAGE)1 in TAE (40 mM Tris
acetate, pH 7.8, 1 mM EDTA) buffer. To determinate the
cleavage sites, samples which had been incubated at 37 °C for 60 min
were analyzed by electrophoresis in 12% polyacrylamide gels containing
7 M urea. For a standard gel shift assay, the reaction
mixtures were incubated on ice for 15 min, and the samples were
analyzed by in 6% PAGE in TAM (40 mM Tris acetate, pH 7.8, 0.5 mM magnesium acetate) buffer. The radioactive materials
were analyzed quantitatively using an image analyzer, Fuji BAS1000.
Potassium Permanganate Footprinting--
Reaction mixtures (50 µl) containing 3 ng of 5' 32P-labeled junction DNA, 25 mM Tris-HCl, pH 8.0, 1 mM MgCl2 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 5%
(v/v) glycerol, and the indicated concentrations of RuvC were incubated
for 10 min at room temperature. Two microliters of 50 mM
KMnO4 was added to the reaction mixtures and incubated for
another 2 min. The reactions were stopped by addition of 2 µl of 14.7 M
-mercaptoethanol. The products were extracted with
phenol, precipitated with ethanol and dried. Pellets were dissolved in
50 µl of 1 M piperidine, and the samples were heated at
90 °C for 30 min. The products were separated by electrophoresis in
12% polyacrylamide gels containing 7 M urea and
analyzed using Fuji BAS1000.
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RESULTS |
DNA Repair Activities of Phe-69 Mutants of RuvC--
RuvC mutants
F69Y, F69W, and F69A were constructed by site-directed mutagenesis of a
plasmid based on pET-8c (24). These mutant proteins and RuvC F69L (23)
were tested for in vivo complementation of the UV repair
deficiency of
ruvC strain HRS1200. RuvC F69W, F69A, and
F69L did not complement the UV sensitivity of HRS1200 (Fig.
1). However, F69Y was active in repair
and near fully complemented the repair defect. The expression level of
these four mutants and wild-type RuvC was determined by Western blot
analysis using anti-RuvC serum (data not shown). All four mutants were
expressed at the same level as wild-type RuvC in HRS1200 cells under
the conditions of the UV sensitivity test. Thus, it is likely that RuvC
F69W, F69A, and F69L do not complement the repair deficiency because
they are deficient in repair and not because they are poorly expressed
or unstable proteins. Because RuvC F69Y complements the
ruvC deficiency but RuvC F69W, F69A, and F69L do not, it is likely that a benzene ring is required at this position of RuvC for
proper enzymatic function.

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Fig. 1.
DNA repair activity of Phe-69 mutants of
RuvC. UV sensitivity test of HRS1200 ( ruvC) cells
harboring ruvC-expression plasmids was performed as
described under "Experimental Procedures." The values shown are the
average of three independent experiments. Plasmids were as follows:
ruvC+ (pRC100, ), vector (pET-8c, ), F69Y
(pRC501, ), F69W (pRC502, ), F69A (pRC503, ) and F69L (pRC504,
previously named pRC226, Ref. 23; ).
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In Vitro Junction Cleavage Activity of RuvC Phe-69
Mutants--
Phe-69 mutant proteins were purified to greater than 99%
homogeneity and characterized in vitro. These proteins were
free of nonspecific nuclease activity (data not shown). RuvC D7N was also used as a negative control in these studies. This mutant has an
amino acid change in the enzyme active site and binds to junction DNA
with the same affinity as wild-type RuvC but is unable to cleave DNA
(21). The cleavage activity of these mutants was examined using a
synthetic Holliday junction, HJ2, as a substrate (21). Fig.
2 shows that RuvC F69Y is partially
active as an endonuclease (63% of wild-type RuvC activity), but RuvC
F69W, F69A, F69L, and D7N do not generate any detectable cleavage
products with the synthetic Holliday junction substrate. During a
longer incubation, F69W cleaved the substrate with low efficiency
(~4% cleavage product), but F69A, F69L, and D7N produced no cleavage product after 2 h (data not shown). Essentially the same results were obtained with another synthetic Holliday junction DNA substrate, X12, which was used previously by van Gool et al. (25),
indicating that the results are not restricted to a particular
substrate. RuvC F69Y cleaved HJ2 and X12 at the same sites as wild-type
RuvC (data not shown). These results confirm the in vivo
results and suggest that Phe-69 can be substituted with tyrosine but
not with other amino acids that lack a benzene ring.

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Fig. 2.
Cleavage of synthetic Holliday junction by
RuvC Phe-69 mutants. Reaction mixtures contained 10 ng of
32P-labeled synthetic Holliday junction HJ2 and 15 ng of
RuvC protein and were incubated for 30 min at 37 °C. The products
were analyzed by 9% PAGE.
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Binding Affinity of Phe-69 Mutant Proteins--
The binding
affinity of the RuvC Phe-69 mutants for a synthetic Holliday junction
was examined using a gel-shift assay in the absence (Fig.
3A) and presence of 100 mM NaCl (Fig. 3B). All RuvC mutants formed a
complex with HJ2 DNA as efficiently as wild-type protein in the absence
of NaCl. This result is consistent with the previous result (23). In
contrast, F69A and F69L did not bind junction DNA in the presence of
100 mM NaCl (Fig. 3B, lanes 11-16). This result
indicates that substitution of Phe-69 by alanine or leucine reduces
binding affinity when electrostatic interaction between RuvC and DNA is
altered by a high concentration of monovalent salt. The fact that RuvC
F69Y retains substantial cleavage activity (Fig. 2) and full binding
activity (Fig. 3) suggests that the aromatic ring of the side chain of
the residue 69 position is involved in a nonelectrostatic interaction
with the Holliday junction DNA substrate and that this interaction is
essential for cleavage by RuvC endonuclease. Interestingly, RuvC F69W
has very low endonuclease activity but binds to the synthetic Holliday
junction as efficiently as or more efficiently than wild-type RuvC even
in the presence of 100 mM NaCl (Fig. 3B,
lanes 8-10). This may indicate that the tryptophan residue
interferes with cleavage of the Holliday junction because it is bulkier
than phenylalanine or tyrosine; nevertheless RuvC F69W binds DNA
similarly to wild-type and F69Y RuvC.

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Fig. 3.
Gel shift assay of binding of RuvC proteins
to the synthetic junction HJ2 in the presence of NaCl at 0 mM (A) or 100 mM (B). Lane 1 contained no protein. Wild-type or mutant RuvC was added as indicated
at increasing concentrations of 10, 20, and 50 nM.
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Aromatic Side Chain of Phe-69 is Involved in Helix Destabilization
at the Holliday Junction Crossover--
The conformation of the
synthetic Holliday junction substrate was previously characterized in
the presence of Mg2+ (26, 27). The protein-free DNA
junction folds into a stacked X-structure, which exhibits 2-fold
symmetry. In this structure, two strands approximate B-form DNA, and
the complementary strands are sharply bent as they pass from one helix
to the other. Base pairing is maintained all around the junction (26,
27). When RuvC binds to the DNA substrate, the crossover is converted
from a folded form into an unfolded form with 2-fold symmetry, and base
pairing around the crossover is disrupted (14, 16). It has been
proposed that disruption of base pairing is a crucial step in forming a
catalytically competent complex between RuvC and the Holliday junction
substrate (14, 16). These observations raise a possibility that Phe-69
is involved in a stacking interaction with a nucleotide base close to
the crossover; formation of the catalytically competent complex may
involve this interaction and the disruption of the base pairing
associated with it. This hypothesis was tested by carrying out
potassium permanganate (KMnO4) analysis of junction DNA
complexes in the presence of wild-type or mutant RuvC (Fig.
4). Potassium permanganate attacks the
C-5=C-6 double bond in thymines that are unstacked, unwound, or
unpaired (28). Thus, this method is useful for detecting base
disruption around a Holliday junction crossover.

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Fig. 4.
Analysis of Holliday junction HJ2L complex
with wild-type and mutant RuvC proteins by potassium permanganate
footprinting. Junction HJ2L with 32P-labeled strand 1 (A) and strand 2 (B) was incubated with the
indicated amount of RuvC in binding buffer. DNA-protein complexes were
treated with permanganate and analyzed by denaturing PAGE as described
under "Experimental Procedures." Lane a shows the ladder
of the Maxam-Gilbert G+A reactions of the labeled strand. The DNA
sequences are shown to the right. In each sequence, the
large bold T is the thymine whose hypersensitivity is
reduced in complexes with RuvC F69A and F69L. The corresponding thymine
bands are labeled with arrows to the right of each
gel.
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A synthetic junction called HJ2L was used in the KMnO4
footprinting studies. HJ2L has longer arms but has the same central 10-base pair sequence as HJ2. HJ2L was used in this experiment, because
it gives a piperidine-cleaved ladder with more similar intervals around
the crossover than HJ2; its use would make it easier to evaluate the
sensitivity of each band of the ladder. Wild-type RuvC cleaves the same
sequences of HJ2 and HJ2L with equal efficiency (data not shown).
Each of the four strands of HJ2L was probed with KMnO4 in
the presence of Mg2+. In the absence of protein, only
background reactivity was observed. In the presence of wild-type RuvC,
all thymine bases within the homologous region and two thymine bases
outside the homologous region were hypersensitive to KMnO4.
The following thymines were highly reactive: three bases in the
homologous region on strand 1 (Fig. 4A, lanes
b-d); the thymine next to the homologous region on strand 2 (Fig.
4B, lanes b-d), and two thymines in the
homologous region on strand 3 (data not shown). Strand 4 has no thymine
bases near the crossover, and no hypersensitive bases were detected on
this strand. A cleavage product was also detected on strand 1 produced
by nicking at 5'-TTT
G-3'. A DNA·RuvC complex with the active site
mutant RuvC D7N produced the same KMnO4 hypersensitivity as
the complex with wild-type RuvC, but no cleavage product was observed
(lanes m and n). The results of these control
experiments were highly consistent with the results of earlier studies
that used a different DNA substrate (16, 29). KMnO4
sensitivity was also examined in complexes between RuvC Phe-69 mutants
and HJ2L. Complexes with F69Y and F69W (lanes e-h) had the
same KMnO4 sensitivity as complexes with wild-type and D7N
mutants of RuvC. In contrast, complexes with RuvC F69A and F69L
demonstrated severely decreased sensitivity at the first thymine base
on strand 1 (Fig. 4A, lanes i-l) and the thymine base on
strand 2 (Fig. 4B, lanes i-l) although very similar
sensitivity was observed for the two thymine bases on strand 3 (data
not shown). These results strongly suggest that the aromatic side chain
of Phe-69 interacts with particular nucleotide(s) and is involved in
inducing an opened state of the crossover upon disruption of the base
pairing. Fig. 5 summarizes the results of
footprinting analysis of RuvC complexes with junction DNA.

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Fig. 5.
Summary of the permanganate footprinting
analysis. Shaded base pairs indicate the 4-base pair
homology core region of HJ2L. Strands 1 and 3 are continuous strands
and strands 2 and 4 are crossover strands (determined by hydroxyradical
footprinting and sensitivity to RuvC-mediated strand cleavage). RuvC
cleavage sites are indicated by vertical arrows. The
crossover point of the junction DNA was assigned as indicated.
Dots indicate the nucleotides hypersensitive to permanganate
when RuvC is bound. The two Ts in large bold letters are the
nucleotides whose hypersensitivity is reduced in complexes with RuvC
F69A and F69L.
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DISCUSSION |
This study presents genetic and biochemical evidence that the
benzene ring of residue Phe-69 in RuvC is catalytically important for
Holliday junction resolution. RuvC F69Y has substantial DNA repair
activity in vivo, and it cleaves synthetic Holliday
junctions with reasonable efficiency in vitro. In contrast,
F69W, F69A, and F69L do not repair DNA damage in vivo or
cleave synthetic Holliday junctions in vitro. RuvC F69A and
F69L show reduced binding to junctions under high salt conditions,
indicating that Phe-69 is involved in the interaction between RuvC and
its DNA substrate. Indeed, the benzene ring of Phe-69 faces the
catalytic center (20), which is a relatively uncommon orientation for
hydrophobic residues such as phenylalanine. RuvC F69W binds DNA
junctions with similar affinity to RuvC wild-type and F69Y, but F69W
can not resolve Holliday junctions in vivo or in
vitro. This suggests that precise interaction of the benzene ring
at position 69 with the junction, that cannot be mediated by a bulky
tryptophan residue, is required for junction resolution. Notably,
orthologs of RuvC from eubacteria have phenylalanine or tyrosine but
not tryptophan at the position equivalent to Phe-69 in E. coli RuvC (22).
Evidence indicates that RuvC binding leads to disruption of base
pairing at the crossover junction. It is likely that this DNA
conformational change is important in the mechanism of cleavage and
resolution by RuvC (14, 16). A prominent wall is located between the
two active centers in the RuvC dimer, and docking studies using RuvC
dimer and junction DNA indicate that this wall disrupts base pairing of
the junction DNA by penetrating the junction center (20). The wall
consists of the protruding loop preceding the second
-helix, and the
aromatic ring of Phe-69 faces the active center. A structure-based
model was proposed based on docking experiments which suggests that the
aromatic ring of Phe-69 makes a stacking interaction with a nucleotide
base close to the junction point (20).
Footprinting analysis with permanganate showed that the DNA substrate
HJ2L contains thymines that become hypersensitive when wild-type RuvC
binds. The hypersensitive bases include 3 thymines in strand 1, a
single thymine in strand 2, and 2 thymines in strand 3 (Fig. 4).
However, RuvC F69A and F69L did not fully induce hypersensitivity at
the first thymine in strand 1 or the thymine in strand 2, whereas the
hypersensitivity of the other thymines was not reduced in complexes
with these RuvC mutants (Figs. 4 and 5). These observations suggest
that Phe-69 is involved in disruption of a particular base pair near
the junction crossover. We propose that the overall disruption of base
pairing is induced when the wall penetrates the junction center (20),
and that the aromatic ring of Phe-69 stabilizes an open structure by a
stacking interaction with a nucleotide base, which is one of the
requirements for catalytic competence. A catalytically important
phenylalanine residue has been reported in the mismatch repair protein
MutS, in which a specific phenylalanine forms a stacking interaction
with a mismatched base (30, 31).
Hydroxyl radical footprinting analysis of the protein-free junction
(data not shown) and identification of the strands cleaved by RuvC
(Fig. 4) revealed structural information on the configuration of the
HJ2L model junction (Fig. 5). Strands 1 and 3 are continuous (noncrossover) strands, and strands 2 and 4 are crossover strands. HJ2L
contains a 4-base pair homologous core in the central region of the
junction (a bimobile junction), which means that it can make two steps
of spontaneous branch migration; thus, HJ2L has three possible junction
points. Recently, we showed that RuvC preferentially nicks the
phosphodiester bond one nucleotide 3' to the crossover point (32). The
cleavage site is 5'-TTT
G-3' in strand 1 (arrow indicates
the cleavage site) and the crossover point is 5'-TT|TG-3'
(vertical bar indicates crossover). These results are shown
schematically in Fig. 5.
The permanganate footprinting analysis using RuvC mutants F69A and F69L
suggest that Phe-69 affects the base pairings 2.5 nucleotides 5' to the
cleavage site (Figs. 4 and 5). It is not possible to determine whether
Phe-69 interacts with the continuous or the crossover strand of the
substrate from the footprinting experiment. However, the model for the
RuvC-junction complex based on docking experiments suggests that the
base on the continuous strand is closer to Phe-69 in each RuvC monomer
than the base in the crossover strand (20). Thus, the data indicate
that each Phe-69 residue in the RuvC dimer forms a stacking interaction with a base 2.5 nucleotides 5' to the cleavage site on the continuous strand. This stacking interaction stabilizes an open state in the
junction core and leads to formation of a catalytically competent protein-DNA complex.
Based on this information, a proposed catalytic mechanism for
RuvC-mediated cleavage is presented in Fig.
6. When RuvC binds the junction DNA, it
induces a change in the DNA configuration from a stacked X structure to
an unfolded core structure (14, 16). The four acidic residues Asp-7,
Glu-66, Asp-138, and Asp-141 constitute the catalytic center of RuvC
(21). These residues coordinate divalent metal cation(s) such as
Mg2+ or Mn2+ and make a pentacoordinated
intermediate using the metal ion(s) and phosphodiester bonds at the
cleavage site on the continuous strand (17, 21, 33). Two basic
residues, Lys-107 and Lys-118, interact with the negatively charged
phosphate of the DNA backbone via electrostatic interactions and
stabilize the pentacoordinated intermediate in which negative charge
accumulates during the transition state of hydrolysis (22). Phe-69
residue forms a stacking interaction with a base 2.5 nucleotides 5' to
the cleavage site to stabilize the open state in the junction core, as
described above. This model is well consistent with the results of
in vivo and in vitro analyses of many mutant RuvC
proteins and with feature of the RuvC structure. Further
physicochemical analyses such as x-ray crystallography of RuvC-Holliday
junction complexes will provide more direct evidence concerning the
mechanism of RuvC-mediated resolution of Holliday junctions.