Lloyd1 1 Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham, UK;2Transgenomic Research Laboratory, Krebs Institute, Department of Molecular Biology and B
Trang 1Holliday junction binding and processing by the RuvA protein
Stuart M Ingleston1, Mark J Dickman2, Jane A Grasby3, David P Hornby2, Gary J Sharples4
and Robert G Lloyd1
1
Institute of Genetics, University of Nottingham, Queen’s Medical Centre, Nottingham, UK;2Transgenomic Research Laboratory, Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK;3Krebs Institute, Centre for Chemical Biology, University of Sheffield, UK;4Department of Biological Sciences, University of Durham, UK
The RuvA, RuvB and RuvC proteins of Escherichia coli act
together to process Holliday junctions formed during
recombination and DNA repair RuvA has a well-defined
DNA binding surface that is sculptured specifically to
accommodate a Holliday junction and allow subsequent
loading of RuvB and RuvC A negatively charged pin
pro-jecting from the centre limits binding of linear duplex DNA
The amino-acid sequences forming the pin are highly
con-served However, in certain Mycoplasma and Ureaplasma
species the structure is extended by four amino acids and two
acidic residues forming a crucial charge barrier are missing
We investigated the significance of these differences by
analysing RuvA from Mycoplasma pneumoniae Gel
retar-dation and surface plasmon resonance assays revealed that
this protein binds Holliday junctions and other branched
DNA structures in a manner similar to E coli RuvA
Sig-nificantly, it binds duplex DNA more readily However it does not support branch migration mediated by E coli RuvB and when bound to junction DNA is unable to pro-vide a platform for stable binding of E coli RuvC It also fails to restore radiation resistance to an E coli ruvA mutant The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA bind-ing, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier They also indicate that such
an obstacle may interfere with the binding of a resolvase Mycoplasmaspecies may therefore process Holliday junc-tions via uncoupled branch migration and resolution reac-tions
Keywords: recombination; DNA repair; RuvABC resolva-some
The formation and subsequent processing of Holliday
junctions are key stages in recombination and DNA repair
that provide the means to repair broken DNA molecules,
generate recombinants in genetic crosses and rescue
repli-cation forks stalled at lesions in the template strands [1–4]
Once formed, these four-way branched DNA structures are
targeted by junction-specific DNA helicases and
endonuc-leases that act, respectively, to move the branch point along
the DNA (branch migration) and to cut specific DNA
strands at or near the crossover, thus releasing duplex DNA
products (resolution) In Escherichia coli, the resolution
reaction appears to be coupled to branch migration via the
formation of a specialized molecular machine composed
of three protein subunits, RuvA, RuvB and RuvC [5,6]
A tetramer of RuvA binds one face of an open Holliday
junction to form a specific complex that supports the
loading of two RuvB ring helicases on opposing duplexes
and of a dimer of RuvC endonuclease on the other face of
the junction in the space between the RuvB rings [7,8] The
RuvAB proteins catalyse junction branch migration while
RuvC resolves the structure to duplex products by
intro-ducing a pair of symmetrically related incisions at specific sequences as the DNA strands move through the complex [8–10]
The RuvA protein plays a pivotal role in processing Holliday junctions It functions as a specificity factor for junction binding, provides a RuvA-junction scaffold for assembly of RuvB and RuvC, and actively participates in the branch migration and resolution reactions [11,12] The atomic structure of RuvA reveals fourL-shaped monomers comprising a fourfold symmetrical platform uniquely adapted for binding four-way branched DNA molecules [13,14] Grooves on the concave surface of the tetramer accommodate each duplex arm of the junction in an open square conformation [13–16] Two helix-hairpin-helix motifs from each monomer make contacts with the phosphodiester backbone on the minor groove side of each duplex arm of the junction [16,17] The junction can be bound by a single tetramer of RuvA [15,16] or enclosed between two tetramers [18] It is not known if binding of a single tetramer of RuvA is sufficient for branch migration
by RuvAB This reaction may require a double tetramer of RuvA or the assembly of a RuvABC resolvasome to anchor the complex [18] In the case of the octameric RuvA complex, one of the tetramers would need to be released to permit loading of RuvC for Holliday junction resolution
At the intersection of the grooves, negatively charged pins consisting of Glu55 and Asp56 from each monomer project towards the centre of the Holliday junction [13,14] The four pairs of negative charges prohibit binding of duplex DNA across the centre of the tetramer and ensure high junction
Correspondence to R G Lloyd, Institute of Genetics, University of
Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK.
Fax: + 0115 9709906, Tel.: + 0115 9709406,
E-mail: bob.lloyd@nottingham.ac.uk
Abbreviations: bio, biotin; SA, streptavidin; RU, response units.
(Received 1 November 2001, revised 3 January 2002, accepted
22 January 2002)
Trang 2specificity [12] Both acidic residues may also participate
directly in the branch migration reaction by forming
water-mediated contacts with unpaired bases [16] Mutations that
alter the charge on these residues stimulate the rate of
branch migration and attenuate the enhanced junction
resolution observed with the RuvABC complex [12]
The negatively charged pin of RuvA is conserved in
almost all bacteria with the exception of three species,
Mycoplasma pneumoniae, M genitalium and Ureaplasma
urealyticum Mycoplasmas belong to the class Mollicutes
and are most closely related to Gram-positive bacteria,
although their genomes have experienced a drastic
com-pression in size In this work we have examined the
properties of M pneumoniae RuvA (MpRuvA) protein to
determine the function of the modified pin The interaction
between MpRuvA and branched DNA substrates and its
inability to form heterologous complexes with E coli Ruv
proteins reveal important differences in junction binding
and processing by Mycoplasma species
M A T E R I A L S A N D M E T H O D S
Strains and plasmids
E coli K-12 strains AB1157 (ruv+), SR2210 (ruvA200),
TNM1208 (DruvAC65 rus-1) have been described
previ-ously [25,29] Strain SI171, a DruvAC65 derivative of BL21
(DE3), was used for overexpression of RuvA proteins [17]
EcRuvA was overexpressed from the pT7-7 construct,
pAM159 [17] The Mycoplasma pneumoniae M129 [30] ruvA
gene was recovered by PCR from genomic DNA obtained
from R Herrmann (Universita¨t Heidelberg, Germany)
Oligonucleotides (5¢-AAACTAAGGCATATGATTGCT
TCAA-3¢ and 5¢-TGCGCCTTATGGATCCGGGACG
CTT-3¢) were designed to amplify the gene and provided
NdeI and BamHI sites (underlined) for cloning the PCR
product in pT7-7 The resulting construct, pSI66, was used
for overexpression of MpRuvA Cells were grown in LB
medium supplemented with ampicillin (50 lgÆmL)1) as
required for maintenance of plasmids Sensitivity to UV
light was measured as described [25]
Protein purification
Purification of MpRuvA followed a similar protocol to
that described for EcRuvA [17,31] RuvB and RuvC
proteins were overexpressed and purified as described
previously [32,33] Protein concentrations were estimated
by a modified Bradford assay using a Bio-Rad assay kit and
bovine serum albumin as standard Amounts of RuvA,
RuvB and RuvC are expressed as moles of the monomeric
protein
DNA substrates
Oligonucleotide synthesis was performed on an Applied
Biosystems 394 DNA synthesiser using cyanoethyl
phos-phoramidite chemistry The biotin phosphos-phoramidite was
obtained from Glen Research DNA substrates were
prepared by annealing appropriate oligonucleotides
follow-ing the procedure described by Parsons [34] The sequence
of oligonucleotides used for the 50 bp junctions J11 and J12,
containing mobile cores of 11 and 12 bp, respectively, have
been described [23,24], as have those for Y junction and linear duplex DNA substrates [20,24] Gel retardation and branch migration assays used substrates in which a single strand had been 5¢ 32P-labelled using [c-32P] ATP and polynucleotide kinase prior to annealing For SPR analysis the following oligonucleotides were used to make a 50-bp static junction, J0, labelled with biotin (bio) at the 5¢ end of one strand: 1 (bio-AAAAATGGGTCAACGTGGGCAA AGATGTCCTAGCAATGTAATCGTCTATGACGTT),
2 (GTCGGATCCTCTAGACAGCTCCATGTTCACTG GCACTGGTAGAATTCGGC), 3 (TGCCGAATTCTA CCAGTGCCAGTGAAGGACATCTTTGCCCACGTTG ACCC), 4 (CAACGTCATAGACGATTACATTG CTAC ATGGAGCTGTCTAGAGGATCCGA) A three-strand junction was made by omitting strand 4 and a 37-bp duplex DNA by annealing oligonucleotides 5 (bio-AATGCTA CAGTATCGTCCGGTCACGTACAACATCCAG) and
6 (CTGGATGTTGTACGTGACCGGACGATACTGT AGCATT)
Gel retardation assays Binding mixtures (20 lL) contained 0.2 ng32P-labelled J11,
Y junction, or linear duplex DNA in 50 mM Tris/HCl
pH 8.0, 5 mM EDTA, 1 mM dithiothreitol, 100 lg/mL BSA and 6% (v/v) glycerol Samples were incubated on ice with RuvA protein for 10 min prior to loading onto a 4% polyacrylamide gel in low ionic strength buffer (6.7 mM Tris/HCl pH 8.0, 3.3 mMsodium acetate, 2 mMEDTA) In RuvAC-junction assays, RuvA was added prior to the addition of RuvC Electrophoresis was at 160 V for 90 min with continuous buffer circulation Gels were dried and analysed by autoradiography and phosphorimaging Branch migration assays
Reaction mixtures (20 lL) contained 0.2 ng of32P-labelled J12 in 20 mM Tris/HCl pH 7.5, 5 mM EDTA, 2 mM dithiothreitol, 100 lgÆmL)1BSA RuvA protein was added before RuvB and reactions incubated at 37°C for 30 min and terminated by the addition of 5 lL of stop mix (2.5% SDS, 200 mM EDTA, 10 mgÆmL)1 proteinase K) with incubation for a further 10 min Reaction products were separated on 10% PAGE in Tris/borate/EDTA buffer (89 mMTris/HCl, pH 8.0, 89 mMborate, 2.5 mMEDTA)
at 160 V for 90 min and analysed as described above Surface plasmon resonance
Surface plasmon resonance was performed using a BIAcore
2000TM (Uppsala, Sweden) Oligonucleotides were diluted
in buffer [10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20] to a final concentration
of 1 ngÆmL)1and passed over a streptavidin (SA) sensor chip at a flow rate of 10 lLÆmin)1until approximately 100–
200 response units (RU) of the oligonucleotide was bound
to the sensor chip surface Proteins were also diluted in Hepes/NaCl/Pi/EDTA/P20 and a range of concentrations (4–8000 nM) were injected over the DNA-charged sensor chip at a flow rate of 20 lLÆmin)1for 3 min and allowed to dissociate for 5 min Bound protein was removed by injecting 10 lL of 1MNaCl This regeneration procedure did not alter the ability of EcRuvA to bind Holliday
Trang 3junction Analysis of the data was performed using
BIA-EVALUATION software To remove the effects of the bulk
refractive index change at the beginning and end of
injections (which occur as a result of a difference in the
composition of the running buffer and the injected protein),
a control sensorgram obtained over the streptavidin surface
was subtracted from each protein injection
Kinetic analysis
The dissociation rate constants were calculated using linear
regression analysis assuming a zero order dissociation using
the equation:
dR=dt ¼ kdR0eÿkd ðtÿt 0 Þ
where dR/dt is the rate of change of the SPR signal, R and
R0, is the response at time t and t0 kdis the dissociation rate
constant
Equilibrium binding analysis
BIAcore equilibrium binding experiments were performed
as described [35] with minor modifications The instrument
was equilibrated at 25°C with 10 mM Hepes, pH 7.4,
150 mMNaCl, 3 mMEDTA, 0.05% (v/v) surfactant p20 at
a flow rate of 100 lLÆmin)1 Baseline data were collected for
45 min at the start of the experiment, before the
incorpor-ation of the protein into the running buffer After
equilib-rium binding profiles had been generated, the responses
from the four flow cells were baseline corrected during the
initial washing phase The response from the reference flow
cell was subtracted from the other three flow cells to correct
for refractive index changes, nonspecific binding and
instrument drift
R E S U L T S
The modified pin structure ofMp RuvA
The negatively charged pin of E coli RuvA (EcRuvA) has
two acidic residues (Glu55 and Asp56) flanked by b sheets
[13,14] This arrangement is conserved in the RuvA
sequences from 45 other bacterial species [12] (Fig 1A and
data not shown), which suggests that the pin architectures
are probably very similar, as demonstrated for
Mycobacte-rium leprae RuvA [18] However, three bacterial species
(M pneumoniae, M genitalium, and Ureaplasma
urealyti-cum) carry RuvA orthologs in which the sequences forming
the pin region differ significantly from this pattern (Fig 1B)
These RuvA proteins have an additional four amino acids
and lack acidic residues at the apex of the intervening loop
Acidic residues that may potentially compensate for the loss
of the negative charge are located nearby in the two
Mycoplasma sequences but are positioned in the region
corresponding to b sheet 6 in the EcRuvA structure [17] The
global structure of the two RuvA proteins would have to be
radically altered to accommodate these residues in the same
position as in EcRuvA In addition, only one acidic residue is
retained in U urealyticum RuvA (Fig 1B) However,
con-servation of sequences in the flanking b sheets suggests that
the general architecture of the pin is probably maintained
Thus, the likely net effect of the altered sequence between b5
and b6 is to produce an extended and uncharged pin
Interaction ofMp RuvA with a Holliday junction
To investigate the effect of these alterations in pin structure
on the DNA binding properties of RuvA we purified the Mycoplasma pneumoniae RuvA protein and compared its activity with that of EcRuvA The protein was overex-pressed in a DruvAC derivative of E coli BL21 (DE3) to prevent contamination with EcRuvA and purified using the procedure devised previously A synthetic Holliday junction containing an 11-bp mobile core was used as a substrate in gel retardation assays to assess the ability of the protein to bind junction DNA Both MpRuvA and EcRuvA bound the junction Each formed two distinct complexes (Fig 2A)
In the case of the E coli protein, the two complexes represent the binding of either a single tetramer of RuvA (complex I) or of two tetramers (complex II) The data presented indicate that MpRuvA has the ability to form similar complexes However, MpRuvA complex II appears less stable as most of the junction is found in complex I (Fig 2A, lanes l–t) In both cases, 100 nMof protein was sufficient to bind all of the junction DNA molecules (Fig 2A, lane j and t) Further quantitative studies revealed that MpRuvA may have a slightly higher affinity for junction DNA than EcRuvA (Fig 2B) The kd values estimated from these data were 18 nMfor MpRuvA and
42 nMfor EcRuvA
Specificity of Holliday junction binding byMp RuvA The E coli RuvA protein targets four-way junctions with high specificity [19,20] Mutations that reduce the net charge
on each subunit result in a significant increase in affinity for duplex DNA [12] We investigated the junction specificity of MpRuvA by analysing its binding to a Y-shaped junction and to linear duplex DNA Like the E coli protein, MpRuvA formed two complexes with a Y junction However, as with the four-way junction, only small amounts of complex II were detected, which again suggests that the loading of two tetramers is less favoured (Fig 3A)
No binding to linear duplex DNA was detected with EcRuvA (Fig 3B, lanes b and c) in keeping with its high selectivity for branched molecules However, traces of two complexes were detected with MpRuvA, even at relatively low concentrations of protein (Fig 3B, lanes d and e)
To analyse the structure specificity of MpRuvA more quantitatively we made use of surface plasmon resonance Biotinylated DNA substrates [a Holliday junction (J0) lacking a homologous core, a three-strand derivative of J0, and duplex DNA] were immobilized on different flow cells
on a streptavidin sensor chip The binding of EcRuvA and MpRuvA to these substrates was examined and the results are shown in Fig 3C,D EcRuvA showed the expected preference for Holliday junction DNA over both three-strand and duplex DNA as evident from the gradient of dissociation illustrated on the sensorgram (Fig 3C) Disso-ciation rate constants were calculated using the equation described in Materials and methods Whilst this equation may not fit the entire range of protein concentrations under all of the experimental conditions described here, it repre-sents the best case scenario, as the analysis is comparative in nature and describes the net stability of the protein:DNA complex The rate constants reveal a three to fourfold difference between the linear duplex/three-strand substrates
Trang 4and Holliday junction bound by EcRuvA (Table 1),
illustrating the additional stability of the Holliday
junc-tion-RuvA complex The binding of the MpRuvA is shown
in Fig 3D and shows little difference in the dissociation rate
constants for the three different DNA-MpRuvA complexes,
demonstrating that these complexes have equal stabilities
Figure 3E shows a direct comparison of the binding of
EcRuvA and MpRuvA to linear duplex DNA and shows
the additional stability of the MpRuvA bound DNA
complex compared to the EcRuvA bound DNA complex
But the results also show an increase in the amount of
MpRuvA binding to duplex DNA compared to EcRuvA,
as indicated by the response (Figs 3C–E) MpRuvA also
formed a complex with a short 24 bp duplex that was not
bound detectably by EcRuvA (data not shown) The SPR
data are broadly consistent with the results obtained from
gel retardation assays confirming that MpRuvA has a reduced specificity for Holliday junctions SPR analysis also shows that the EcRuvA and MpRuvA bind to the DNA with fast association rate constants (ka) This results in mathematical models that poorly fit the data, and calcula-tions using kaand kdto obtain the equilibrium dissociation constant would be erroneous
Equilibrium binding analysis was performed to further analyse the interaction of MpRuvA with Holliday junction and duplex DNA (Fig 4) RuvA protein was placed directly
in the running buffer and continually passed over the sensor chip surface containing duplex or Holliday junction attached to different flow cells The binding profile of the MpRuvA interaction with these DNA substrates is shown
in Fig 4A The sensorgram reveals that MpRuvA protein, like EcRuvA (Fig 4B), binds with high affinity to the
Fig 1 The modified pin structure of MpRuvA (A) Structure of the EcRuvA-Holliday junction DNA complex [15].
A tetramer of RuvA (opposing monomers are in shades of grey) binds the Holliday junction in an open square conformation The duplex arms of the junction are bound
in grooves on the concave surface of the protein and converge at a centrally located pin structure formed by Glu55 and Asp56 (red) in each RuvA subunit (B) Alignment
of bacterial RuvA proteins showing conservation of the pin region Residues 42–65 of EcRuvA are aligned with homologous sequences from selected bacterial species Residues conserved in the majority of RuvA sequences from 46 bacteria (data not shown) are highlighted in bold Arrows denote the position of b sheets
5 and 6 in the EcRuvA structure [14,17] Acidic pin residues are highlighted in red, as are negatively charged residues located nearby in the RuvA sequences from
M pneumoniae, M genitalium, and
U urealyticum.
Trang 5Holliday junction at relatively low concentrations of protein
(0.112 and 1.12 nM) Binding to duplex DNA is not
observed until a concentration of 11.2 nM is passed over
the sensor chip surface (Fig 4A) Significantly, these results
reveal that MpRuvA has a higher affinity for duplex DNA
than the EcRuvA protein Binding of EcRuvA to duplex
DNA is not evident until a concentration of 90.4 nM is
reached (Fig 4B) Thus MpRuvA bound to the duplex at a
10-fold lower concentration and assuming the mechanism
and mode of binding is the same, the MpRuvA has a 10-fold
higher affinity for duplex DNA Despite this difference,
MpRuvA retains Holliday junction specificity with similar
kinetics and stoichiometry as EcRuvA
Mp RuvA is unable to interact with E coli RuvB
and RuvC proteins
RuvA and RuvB mediate the branch migration of Holliday
junctions and in vitro promote the dissociation of synthetic
junction substrates to yield flayed duplex products [19] We
examined MpRuvA to see if it could form a branch
migration complex with E coli RuvB Heterologous branch
migration activity has previously been demonstrated using
M lepraeRuvA with E coli RuvB [21] and E coli RuvA
with Thermus thermophilus RuvB [22] MpRuvA was
incubated with E coli RuvB and synthetic Holliday
junc-tion J12 in reacjunc-tions containing Mg2+and ATP (Fig 5A,
lanes j–p) In contrast to reactions containing EcRuvA
(Fig 5A, lanes b–h), no unwinding of the synthetic Holliday
junction was detected in reactions containing MpRuvA
Similar results were obtained using other junctions differing
in sequence and length of mobile core (data not shown) The
results indicate that MpRuvA is unable to form a functional
branch migration complex with E coli RuvB
The coupling of branch migration and resolution medi-ated by the E coli RuvABC resolvasome complex requires the binding of RuvA to one face of the junction and RuvC
to the other [8,9] Complexes formed by the loading of both RuvA and RuvC on a synthetic junction can be detected using a gel retardation assay [23] We used such an assay to
Fig 2 Holliday junction binding by MpRuvA (A) Gel retardation
assay showing the formation of complexes I and II with junction J11.
Binding mixtures contained 0.2 ng32P-labelled J11 DNA and 0, 0.1,
0.5, 1, 2, 5, 10, 20, 50, and 100 n M of EcRuvA (lanes a–j) or MpRuvA
(lanes k–t) proteins (B) Titration of MpRuvA and EcRuvA showing
the relative binding of J11 Values are the mean of two independent
experiments and are based on the fraction of the total DNA bound.
Fig 3 Interaction of MpRuvA with branched DNA structures and lin-ear duplex molecules (A) Gel retardation assay showing binding of RuvA proteins to a Y-junction DNA substrate Reactions contained 0.2 ng 32 P-labelled DNA and RuvA at 2 n M (lanes b and d) or 20 n M
(lanes c and e) (B) Gel retardation assay showing binding of RuvA proteins to linear duplex DNA Reactions contained 0.2 ng
32 P-labelled DNA and RuvA at 10 n M (lanes b and d) or 100 n M (lanes
c and e) (C) Surface plasmon resonance sensorgram showing binding
of EcRuvA (8 l M ) to duplex, three-strand and Holliday junction DNA (D) Surface plasmon resonance sensorgram showing binding of MpRuvA (6.4 l M ) to duplex, three-strand and J0 DNA (E) Surface plasmon resonance sensogram showing the binding of EcRuvA (6 l M ) and MpRuvA (4 l M ) to duplex DNA.
Trang 6investigate whether E coli RuvC could bind a junction
already bound by MpRuvA With 200 nMRuvC and low
concentrations of EcRuvA, a RuvA/junction/RuvC
com-plex was visualized (Fig 5B, lanes c and d) No such
complex could be detected using MpRuvA (Fig 5B, lanes
l–r) The only complexes seen were those formed by the
binding of RuvC alone or of a double tetramer of MpRuvA
(complex II) The absence of MpRuvA complex I may be
significant, especially as this is the predominant complex
formed in the absence of RuvC (Fig 2A) It is possible that
such complexes do bind RuvC but that such binding
destabilizes the RuvA–junction interaction
Effect ofMp RuvA on DNA repair in E coli ruv mutants
The ability of MpRuvA protein to promote DNA repair
in vivowas investigated by introducing plasmid constructs
encoding MpRuvA or EcRuvA into E coli strains SR2210 (ruvA200) and the ruv+ control, AB1157 The plasmid expressing MpRuvA (pSI66) failed to improve the UV sensitivity of the ruvA mutant SR2210 (Fig 6A), which is not surprising given that MpRuvA fails to form productive interactions with E coli RuvB or RuvC Indeed, survival was actually reduced This negative effect is most likely due
to MpRuvA blocking the access of other junction process-ing enzymes such as RecG [24] Expression of MpRuvA also reduced survival of the ruv+ AB1157 strain (Fig 6B) However, the effect was rather modest and we conclude that overexpression of MpRuvA does not interfere significantly with junction processing by the resident E coli RuvABC system
To further investigate the ability of MpRuvA to block junction processing in vivo, we made use of strain TNM1208 (DruvAC rus-1) This strain lacks the RuvABC resolution pathway due to deletion of the ruvA and ruvC genes However, it is resistant to UV light because the rus-1 mutation activates an alternative resolvase (RusA) that is able to process Holliday junctions very efficiently in the
Table 1 Dissociation rates for MpRuvA and EcRuvA-DNA complexes.
DNA
Dissociation rate constant (k d ) (1/s) ± SDa
Holliday junction 6.1 · 10)4± 2.2 · 10)5 5.5 · 10)4± 4.2 · 10)5 Three-strand junction 7.4 · 10)4± 3.0 · 10)5 17 · 10)4± 1.9 · 10)4 Duplex 6.2 · 10)4± 2.3 · 10)5 19 · 10)4± 2.2 · 10)4
a Determined using surface plasmon resonance analysis.
Fig 4 Equilibrium binding profiles of MpRuvA and EcRuvA on
Holl-iday junction (J0) and linear duplex DNA substrates (A) MpRuvA was
incorporated in the running buffer at concentrations of 0.0112 n M (a),
0.112 n M (b), 1.12 n M (c) and 11.2 n M (d) (B) EcRuvA was
incor-porated in the running buffer at concentrations of 0.00904 n M (a),
0.0904 n M (b), 0.904 n M (c), 9.04 n M (d) and 90.4 n M (e) The arrows
indicate the time at which the concentration of the protein was altered.
Fig 5 Interactions between RuvA and either RuvB or RuvC (A) Branch migration assay showing the dissociation of Holliday junction to flayed duplex products Reactions contained 0.2 ng 32
P-labelled J12 DNA and proteins as indicated (B) Gel retardation assay showing the formation of RuvAC-junction complexes Binding mixes contained 0.2 ng 32 P-labelled J12 DNA and proteins as indi-cated.
Trang 7absence of RuvABC [25–27] The introduction of a plasmid
expressing EcRuvA into this strain increases sensitivity to
UV light (Fig 6C), presumably by blocking Holliday
junction resolution by RusA [17] The plasmid encoding
MpRuvA also increases sensitivity to UV, but the effect is
considerably less severe (Fig 6C) This finding suggests that
MpRuvA is less able to inhibit the processing of Holliday
junctions in vivo than EcRuvA despite the fact that both
bind synthetic Holliday junctions with similar affinities
in vitro(Fig 2A)
D I S C U S S I O N
The negatively charged central pin on the DNA binding
surface of RuvA plays a crucial role in junction targeting
and processing It constrains the rate of branch migration
by RuvAB and influences resolution by RuvABC [12] The
importance of this structure is reflected in the high
conservation of the sequences forming the pin in the
majority of bacteria with the exception of two Mycoplasma
species and one of Ureaplasma In the RuvA proteins from
these organisms the pin sequence is extended by four
residues and lacks negatively charged residues at the apex of
the structure We investigated the properties of the RuvA
protein from M pneumoniae to see how these modifications
affected its interaction with DNA
The MpRuvA protein bound the four-way branched
Holliday junction structure with a high affinity However,
relative to EcRuvA, it displayed an increased affinity for
Y-shaped duplex DNA structures, three-strand junctions
and linear duplex DNA Its affinity for linear duplex DNA
is approximately 10-fold higher than the E coli protein The
results suggest that the modified pin influences the ability to
bind duplex DNA and is consistent with observations by
Ingleston et al [12] showing that mutations in EcRuvA that
reduce the net negative charge on the pin, or which add
positive charges, result in an increase in binding to duplex
DNA
As with the E coli protein, we found that a synthetic
Holliday junction can bind either one or two tetramers of
MpRuvA However the octameric complex (complex II) appears less stable than that formed with EcRuvA As the pin region of MpRuvA contains an additional four amino acids it is likely that the pin is extended and this extension could cause steric clashes across the central cavity of the open Holliday junction that interfere with stable binding
of a tetramer on both faces The reduced stability of the octamer complex may explain the modest negative effect
of MpRuvA compared with EcRuvA on DNA repair mediated by the RusA resolvase in strain TNM1208 (Fig 6C) This protein forms a very stable octameric complex and when overexpressed is therefore much more likely to prevent RusA gaining access to a Holliday junction Single tetramers of EcRuvA and MpRuvA bind junction DNA with similar affinities However, such complexes are less likely to inhibit RusA as one face of the junction would remain free of protein and this may be sufficient for RusA to load on the DNA and resolve the structure
We found that MpRuvA is unable to promote DNA repair in E coli ruvA mutants This is most likely a consequence of its failure to assemble a functional branch migration complex with E coli RuvB Certain conserved residues in domain III of EcRuvA (Leu167, Leu170, Tyr172 and Leu199) are known to participate in protein–protein interactions with EcRuvB [11,14] MpRuvA has the first three of these residues but differs in the replacement of Leu199 with isoleucine It is possible that this subtle change accounts for the inactivity of the hybrid MpRuvA-EcRuvB branch migration motor, although other differences affect-ing the architecture of MpRuvA domain III cannot be excluded Mycobacterium leprae RuvA, which retains a conserved leucine at this position, forms an active branch migration complex with EcRuvB [21] Compensatory changes in the MpRuvB sequence should correspond to the alterations in MpRuvA that prevent heterologous contacts with EcRuvB Isoleucine residues at positions 148 and 150 in EcRuvB are critical for the formation of complexes with EcRuvA [28] In MpRuvB these amino acids are replaced by the alternative hydrophobic residues, valine and methionine, respectively These substitutions at the MpRuvA–RuvB interface are likely to be responsible for blocking the formation of functional complexes between MpRuvA and EcRuvB
We also found that E coli RuvC was unable to form a complex with a junction already bound by MpRuvA, at least not one stable enough to be detected in a gel retardation assay In common with other Gram-positive bacteria, M pneumoniae lacks a homologue of RuvC [6] It
is therefore possible that branch migration and resolution are uncoupled in these species [18] The assembly of a RuvABC complex is necessary for efficient resolution of Holliday junctions in E coli and presumably imposes constraints on the evolution of each Ruv protein In particular, RuvA may have to maintain a compact acidic pin that does not project at the junction core so that the conformation of the RuvA-bound junction allows stable loading of RuvC In the absence of a RuvC, the constraints
on MpRuvA would be reduced and limited to those factors necessary for junction binding and the loading of RuvB However, several Gram-positive bacteria that lack RuvC apparently retain the conserved pin architecture of EcRuvA [6,12] In fact, M pulmonis RuvA has a pin that more
Fig 6 Survival of UV-irradiated Escherichia coli strains carrying
plasmids expressing either MpRuvA or EcRuvA proteins (A) Strain
SR2210 (ruvA200) (B) Strain AB1157 (ruv + ) (C) Strain TNM1208
(ruvAC rus-1) The plasmid constructs used are identified in (B) Values
are the mean of at least two independent experiments.
Trang 8closely resembles the standard pattern rather than its closely
related Mollicutes (Fig 1B) In addition, M leprae RuvA,
which has an apparently identical pin to EcRuvA, also fails
to form junction complexes with EcRuvC in a gel
retarda-tion assay, perhaps suggesting that there are stabilizing
contacts across the junction that are independent of pin
structure [21] Clearly there are subtle differences in the way
Holliday junctions are processed by Mycoplasmas Further
insights into the mechanism of Holliday junction branch
migration and resolution await the identification and
characterization of the novel resolvase employed in
Gram-positive eubacteria
A C K N O W L E D G E M E N T S
We thank Richard Herrmann for Mycoplasma pneumoniae genomic
DNA This work was supported by grants from the Biotechnology and
Biological Sciences Research Council, the Wellcome Trust, the Royal
Society, and the Medical Research Council M J D was in receipt of a
Prize Studentship from the Wellcome Trust.
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