From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India
Received for publication, January 16, 2001, and in revised form, February 22, 2001
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ABSTRACT |
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Uracil, a promutagenic base in DNA can arise by
spontaneous deamination of cytosine or incorporation of dUMP by DNA
polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG),
the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded
DNA binding protein (SSB) facilitated uracil excision from certain
structured substrates by E. coli UDG (EcoUDG)
and suggested the existence of interaction between SSB and UDG. In this
study, we have made use of the chimeric proteins obtained by fusion of
N- and C-terminal domains of SSBs from E. coli and
Mycobacterium tuberculosis to investigate interactions
between SSBs and UDGs. The EcoSSB or a chimera containing
its C-terminal domain interacts with EcoUDG in a binary
(SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera
containing the N-terminal domain from EcoSSB showed no
interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for
interaction with EcoUDG. The data also suggest that the
C-terminal domain (34 amino acids) of MtuSSB is a
predominant determinant for mediating its interaction with
MtuUDG. The mechanism of how the interactions between SSB
and UDG could be important in uracil excision repair pathway has been discussed.
Uracil residues appear in DNA as a result either of spontaneous
deamination of cytosine or of misincorporation of dUMP by DNA
polymerase. Uracil DNA glycosylase
(UDG)1 excises uracil
residues from DNA and prevents mutations from arising to maintain
genomic integrity. The UDG-directed base excision repair pathway
has been shown to involve at least five proteins, UDG, AP endonuclease
IV, RecJ, DNA polymerase I, and DNA ligase, and to utilize the single
nucleotide gap-filling activity (1). The highly inefficient excision of
uracil from structured substrates led to a proposal that melting of
such structures may facilitate efficient repair (2). We subsequently
showed that the inclusion of Escherichia coli SSB
(EcoSSB) in the reactions augmented uracil release from the
structured substrates by E. coli UDG (EcoUDG; Ref. 3). The KMnO4 footprint and Tm
analyses showed that SSB melted the hairpin substrates (3, 4).
Recently, we cloned and purified SSB from Mycobacterium
tuberculosis (MtuSSB; Ref. 5). Its biochemical
characterization revealed that it is similar to EcoSSB in
its oligomerization status and various DNA binding properties
(5). Surprisingly, inclusion of MtuSSB in the reactions led
to a decrease in uracil excision by EcoUDG (4). Thus, it
appeared that the effect of SSB on uracil release from a structured
substrate by EcoUDG was not merely a consequence of melting
of the structured substrate. The binary (SSB-UDG) or the ternary
interaction (DNA-SSB-UDG) may also influence the activity of UDG.
Single-stranded DNA-binding proteins (SSBs) are members of an important
class of proteins that play an essential role in various DNA
transactions (6). EcoSSB, the archetype of the prokaryotic SSBs, is a homotetramer consisting of monomeric subunits of 177 amino
acids (7). The secondary structure prediction suggests that
EcoSSB can be divided into two parts, an N-terminal domain (~120 amino acids) rich in The human homolog of single-stranded DNA-binding protein, replication
protein A, was shown to interact with XPF-ERCC1 and participate
in the nucleotide excision repair pathway (12). More recently,
replication protein A has been shown to interact with human UDG and
participate in the uracil excision repair pathway (13, 14). However,
there are no reports wherein the prokaryotic SSBs have been shown to
interact with UDGs or any other DNA glycosylases.
M. tuberculosis continues to be the pathogen that causes
most casualties worldwide. To develop new means to control this
pathogen, it is important to understand the biology of this organism.
Because of the high G+C contents of its genome (15) and the habitat of
the host macrophages where the bacterium resides, cytosine deamination
may constitute a major form of DNA damage in these organisms, making
UDG a crucial DNA repair enzyme. In this report, we have studied the
interaction between UDGs and SSBs from E. coli and M. tuberculosis, and we show that the C-terminal domain of SSB
mediates interaction with UDG, both in the absence and presence of DNA.
Purification of SSBs and UDGs--
The native and the
chimeric SSBs (EcoSSB, MtuSSB,
MtuEcoSSB, and EcoMtuSSB) were purified by the
method already described (5, 16). Purification of EcoUDG and
MtuUDG was carried out as described previously (4, 17).
Purified proteins were estimated by modified Bradford's dye binding
assay using bovine serum albumin as a standard (18), analyzed by
electrophoresis on 15% polyacrylamide gels containing 0.1% SDS under
reducing conditions, and visualized by Coomassie Brilliant Blue R-250
staining (19).
Uracil DNA Glycosylase Reactions--
10 pmol of a hairpin
substrate, Loop-U2, containing uracil in the second position of the
tetra loop, 5'-CTAGAGGATCCTUTTGGATCCT-3', was 5' end-labeled using
[ Association of the UDG Activity with SSBs--
Loop-U2 (1 pmol,
20,000 cpm) was incubated with 5 pmol of SSBs or ribosome recycling
factor (EcoRRF)2
in UDG buffer for 10 min at 37 °C. The reaction was terminated with
5 µl of 0.2 N NaOH and processed for the detection of the uracil excision activity as above.
Yeast Two-hybrid Assay System--
The yeast two-hybrid analyses
were performed using the HIS3 and Electrophoretic Mobility Supershift Assays (EMSA)--
The SSBs
(10 pmol) were incubated with 1 pmol (20,000 cpm) of a 27-mer
single-stranded oligomer, 5'-CACCTGTATCATATTCGTCGGCGAGCT-3' (16), with
or without UDG (20 pmol) for 30 min in the binding buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5% glycerol
(v/v), 50 µg/ml bovine serum albumin) and separated on a native
polyacrylamide (8%) gel (30:0.5, acrylamide:bisacrylamide) using 0.5×
Tris borate-Na2EDTA buffer for 1-2 h at 150 V (~8
V·cm Binding of Chimeric SSBs to Loop-U2--
Loop-U2 (1 pmol, 2 × 105 cpm) was incubated with MtuEcoSSB or
EcoMtuSSB (0.1, 0.5, 1, 2.5, 5.0, 7.5, 10, or 15 pmol of the tetramer) in the binding buffer and analyzed by native polyacrylamide gels as in EMSA detailed above. The SSB-DNA complex and free DNA were
quantified on a Bioimage Analyzer (Fuji), and the percentage of
complex formed was plotted against the amounts of SSB in the reaction.
Far Western Blot Analysis--
Proteins
(MtuSSB, EcoSSB,
MtuEcoSSB, EcoMtuSSB,
EcoRRF, EcoUDG, and MtuUDG) were
electrophoresed on 15% polyacrylamide gels containing 0.1% SDS under
reducing conditions and transferred to a polyvinylidene difluoride
membrane (0.45 µm; Amersham Pharmacia Biotech) by electroblotting
(22). The transfer was carried out for 3 h at 200 mA. The
membranes were blocked overnight with 1% bovine serum albumin in TBS
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl), washed
with TBS, and overlaid with EcoUDG or MtuUDG (100 µg) in 7 ml of TBS for 2 h. After washing three times with TBS,
the membranes were incubated with antibodies against EcoUDG
or MtuUDG (1:2, 500 dilution) for 1 h at room
temperature. After thorough washing, the blots were incubated with
alkaline phosphatase-conjugated goat anti-rabbit IgG (Life
Technologies, Inc.) at a 1:2000 dilution for 45 min at room
temperature, washed three times with TBS, and developed with 12 µM p-nitroblue tetrazolium chloride and 6 µM 5-bromo-4-chloro-3-indolyl phosphate in 0.15 M Tris-HCl, pH 9.5, and 5 mM MgCl2
(23).
Surface Plasmon Resonance Studies--
Experiments were
performed exactly as described (4). The 5'-biotinylated single-stranded
oligomer was immobilized on a SA5 sensor chip, and the various SSBs
(EcoSSB, MtuSSB, MtuEcoSSB, and
EcoMtuSSB) were passed over the surface to obtain a stable DNA-SSB platform. Thereafter, different concentrations of the EcoUDG and the MtuUDG (400-10,500
nM) were serially passed over the DNA-SSB surface using HBS
buffer (10 mM Hepes, pH 7.4, 50 mM NaCl, 3.4 mM Na2EDTA, 0.005% Tween 20). The increase in
the response units indicated the interaction between the UDGs and the
DNA-SSB complex. At the end of each injection of UDG, there was a fall
in the response units, which indicated the dissociation of UDGs from
the DNA-SSB surface. The sensograms thus obtained were utilized to
evaluate the kinetic and equilibrium parameters using the BIAevaluation software.
Association of UDG Activity with SSBs--
In our earlier studies,
we purified SSBs from E. coli BW310, an
ung Yeast Two-hybrid Assays--
The yeast two-hybrid approach was
used to determine whether the SSBs and the UDGs are capable of
interacting in vivo; this was carried out exactly as
described earlier (16). As expected from the homotetrameric nature of
the EcoSSB and MtuSSB, the yeast transformants
harboring SSBs fused to the activation and the binding domain of GAL4
show growth on medium lacking histidine, tryptophan, and leucine and
containing 10 mM 3-aminotriazole (Fig.
2, sectors 7 and 8,
respectively). The Saccharomyces cerevisiae HF7c alone or
transformants harboring EcoUDG fused to the activation
and the binding domains do not grow under the same conditions (Fig. 2,
sectors 1 and 5, respectively). The transformants
harboring EcoSSB or MtuSSB in combination with
EcoUDG or MtuUDG showed growth (Fig. 2,
sectors 2-4 and 6). These data show that
in vivo, SSBs interact with UDGs in homologous
(EcoSSB with EcoUDG or MtuSSB with
MtuUDG) and heterologous (EcoSSB with
MtuUDG or MtuSSB with EcoUDG)
combinations. The Far Western Analyses--
The sequence comparison of
EcoSSB and MtuSSB showed maximum variation in
their C-terminal domains downstream of amino acid position 129 (EcoSSB numbering; Refs. 5 and 16). Because the C-terminal
region of EcoSSB has been implicated in its in vivo function, we generated two chimeric constructs between the N-
and C-terminal domains of the two SSBs. The DNA sequence at amino acid
position 129 (EcoSSB numbering) contains a BamHI
site in MtuSSB. As a first step in generating the chimeric
constructs, the BamHI site was introduced into the
EcoSSB gene by site-directed mutagenesis (G129S mutation),
and then the DNA sequences corresponding to the N- and C-terminal
domains were swapped between the two SSBs (16) (Fig.
3). The construct MtuEcoSSB
contains the N-terminal portion from MtuSSB and the
C-terminal domain (48 amino acids) from EcoSSB. Similarly,
the chimeric construct EcoMtuSSB comprises the N-terminal
domain from EcoSSB and the C-terminal domain (34 amino
acids) from MtuSSB. These chimeric SSBs formed homotetramers and bound DNA akin to their native counterparts (16). The native and
the chimeric SSBs and EcoRRF were purified (Fig.
4A) and used in a far Western
analysis. The interaction of EcoUDG or MtuUDG with the SSBs was detected by respective polyclonal anti-UDG antibodies (Fig. 4, B-E). Positive controls (EcoUDG and
MtuUDG) in lanes 6 (Fig. 4, B and
C, respectively) showed that both of the polyclonal antibodies detected UDGs with similar efficiencies. Further, as expected, neither of the antibodies cross-reacted with
EcoRRF, which was used as a negative control (Fig. 4,
B-E, lanes 5). The membrane probed by
anti-EcoUDG antibodies revealed that EcoUDG interacted with both EcoSSB and MtuEcoSSB (Fig.
4B, lanes 2 and 3) but not with
MtuSSB and EcoMtuSSB (Fig. 4B,
lanes 1 and 4). Similarly, MtuUDG
interacted with MtuSSB and EcoMtuSSB (Fig.
4C, lanes 1 and 4) but not with
EcoSSB and MtuEcoSSB (Fig. 4C,
lanes 2 and 3). These observations suggest that
the UDGs and SSBs from homologous sources (belonging to the same
organism) interact with each other. Because the UDGs also interacted
with the chimeric SSBs containing C-terminal domains from SSB of the
homologous source, these observations suggest that the interaction
between UDG and the SSB is mediated through the C-terminal domain of
the latter. When the amounts of SSBs bound to the blots were increased (Fig. 4, D and E), the interactions between the
heterologous combinations such as those of EcoUDG with
MtuSSB (Fig. 4D, lane 1) and
MtuUDG with EcoSSB and MtuEcoSSB could
also be discerned (Fig. 4E, lanes 2 and
3), suggesting that the nature of their interactions is weak. Nevertheless, these interactions appear to be specific, because
no interactions of UDGs with EcoRRF were detected (Fig. 4,
D and E, lanes 5).
Electrophoretic Mobility Supershift Assays--
The experiments
described thus far showed binary interactions between SSBs and UDGs. To
investigate the possibility of ternary interactions involving DNA, SSB,
and UDG, electrophoretic mobility shift/supershift assays were carried
out. As expected, EcoSSB, MtuSSB,
MtuEcoSSB, and EcoMtuSSB formed complexes with
DNA (Fig. 5A, compare
lanes 2 and 5 with lanes 1 and
4, and Fig. 5B, compare lanes 2 and
4 with lane 1). Further, consistent with the
observations of the far Western analysis (Fig. 4B), when
EcoUDG was added to DNA-EcoSSB or
DNA-MtuEcoSSB complexes, a further shift was observed (Fig.
5A, lanes 3 and 6). However, under the
same conditions, we failed to detect an interaction between
MtuSSB or EcoMtuSSB and EcoUDG (Fig.
5B, lanes 3 and 5), corroborating our
earlier finding of the far Western analysis (Fig. 4B,
lanes 1 and 4). Thus, as was the case with binary
interactions, transplantation of the C-terminal 48 amino acids of
EcoSSB into the corresponding region of the
MtuSSB was sufficient for a specific ternary interaction. However, when we performed a similar analysis utilizing
MtuUDG with the SSBs, a supershift over and above the
SSB-DNA complex could not be detected (data not shown). A possible
reason for the lack of supershift by MtuUDG could be its
high pI (9.5 as opposed to 6.6 of EcoUDG). In fact, when we
performed a Western blot analysis of the native gels used for EMSA
using polyclonal antibodies to MtuUDG, MtuUDG was
found lodged in the wells (data not shown).
SPR Studies--
We have earlier used this methodology to detect
interaction among the ternary complexes (DNA, SSBs, and UDGs) (4). We
showed that the homologous combinations of SSBs and UDGs
(EcoSSB with EcoUDG and MtuSSB with
MtuUDG) show stronger affinity, (KD, 1.7 × 10
Use of chimeric SSBs in similar experiments shows that the
MtuEcoSSB interacted with both the EcoUDG and
MtuUDG but that the strength of these interactions
(KD, 2.44 × 10 Effect of Chimeric SSBs on Uracil Excision by UDGs--
We earlier
showed that EcoSSB enhanced excision of uracil from Loop-U2
by EcoUDG and MtuUDG. On the contrary, although
the inclusion of MtuSSB in the reactions containing the same
substrate with MtuUDG stimulated uracil excision, it
resulted in decreased activity of EcoUDG, most likely as a
consequence of inappropriate interaction (4). Far Western analysis and
the SPR studies showed a discernible interaction between the chimeric
SSBs and UDGs (for most of the combinations). Therefore, it was of
interest to investigate the effect of the chimeric SSBs on uracil
excision activity of the UDGs using Loop-U2 as a substrate.
Neither MtuEcoSSB nor EcoMtuSSB alone showed any
uracil excision activity (Fig.
6A, lanes 1 and
2). However, when included in the reactions along with
EcoUDG, MtuEcoSSB stimulated the uracil excision
activity (Fig. 6A, compare lanes 4 and
5 with lanes 7 and 8), and
EcoMtuSSB showed a decrease in uracil excision activity of
EcoUDG (compare lanes 4 and 5 with
lanes 10 and 11). Furthermore, as observed before
with the native SSBs, the chimeric SSBs led to a stimulation of uracil
excision by MtuUDG (Fig. 6B, compare lane
3 with lanes 6 and 9).
Thus, these results suggest that the C-terminal domains within the
chimeric SSBs are sufficient to mimic the typical effects of their
native counterparts on the uracil excision activity of the two UDGs
from Loop-U2. However, not surprisingly, the effects of chimeric SSBs,
which contain the C-terminal domains in the context of heterologous
N-terminal domains, on uracil excision are not as pronounced as seen
before with the native SSBs (3). Analysis of the DNA binding activity
of the chimeric SSBs revealed that they both bound Loop-U2 with similar
efficiency (Fig. 7), suggesting that, as
seen earlier with the mutants of EcoSSB deleted for the
C-terminal domain (11), the DNA binding properties of SSBs can be
mediated by the N-terminal domains, independent of the C-terminal
domains. However, for the functional participation of the C-terminal
domain with various cellular proteins, the context of the N-terminal
domain is also important. Occurrence of such "cross-talk" between
the N- and C-terminal domains of SSB could explain why in our earlier
studies, the MtuEcoSSB chimera, despite containing the
C-terminal domain from EcoSSB, failed to complement a
SSBs play a vital role in a variety of DNA metabolic processes
including replication, recombination, and repair (6). There is
increasing evidence for the association of SSBs with several proteins
such as the Recent studies with the human UDG have shown that it interacts with the
34-kDa subunit and the trimeric form of the human homolog of the
single-stranded DNA-binding protein, replication protein A (13).
Subsequently it was demonstrated that UDG also interacts with
proliferating cell nuclear antigen, suggesting that these
proteins exist as a part of a multiprotein complex that carries out the
long patch base excision repair involving proliferating cell
nuclear antigen, polymerase Thus, it appears that the SSB-UDG interaction would be important in
uracil excision repair in the regions of the genome that become
transiently single-stranded, such as the transcription bubble or
replication fork. It is not surprising, therefore, that in humans,
expression of UDG is cell cycle-regulated (31). From a physiological
perspective, it would be preferable to have noninstructional lesions
(AP sites) than the miscoding bases in the DNA during replication,
because such lesions allow for postreplicative repair to occur. A DNA
glycosylase could potentially alter a miscoding base into an AP site if
it was associated with the protein complexes at the replication fork
(32). Further, detection and removal of uracil from the newly
synthesized DNA at the replication fork will also be ideal in avoiding
the consequences of its misincorporation by the DNA polymerases.
The single-stranded regions in DNA are inherently prone to cytosine
deamination. Because there is a greater potential of extrusion of DNA
into complex structures in the single-stranded regions that may arise
in the DNA as a consequence of various physiological processes, our
studies on the uracil release from the hairpin DNA (3, 33, 34) and the
effect of SSB on UDG activity on such substrates provide a good model
system to examine the uracil excision repair pathway. We have earlier
shown that one aspect of SSB-mediated stimulation of base excision
repair is destabilization of the structured substrates (3, 4). The
SSB-UDG interactions that we demonstrate here could also be crucial
from the physiological perspective. The observation that UDG can
interact with the SSB in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG)
complex suggests that SSB may facilitate recruitment of UDG to the site
of the lesion by serving as a carrier. Alternatively, by virtue of
the ternary interactions, the DNA-bound SSB may enhance the binding of
the UDG to the site of the lesion. Further, the mechanism of how SSB
switches its interacting partners including UDG, most of which localize
to the C-terminal domain, remains to be clarified.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices and
-sheets and a
C-terminal domain (~60 amino acids) having no defined structure (8,
9). Studies with EcoSSB N-terminal fragments obtained by
cleavage at Arg115 by trypsin (SSBT) or
at Trp135 by chymotrypsin (SSBC) have
established that the N-terminal domain is responsible for
tetramerization and DNA binding (10). The C-terminal region has been
suggested to be important in the interaction of SSBs with various
proteins in E. coli (11).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and purified using Sephadex G-50 minicolumns
(2, 3, 20). For assays, 1 pmol of Loop-U2 (20,000 cpm) was incubated in
the presence or absence of 5 pmol of the various SSB tetramers in UDG
buffer (50 mM Tris-HCl, pH 8.0, 1 mM
Na2EDTA, 1 mM dithiothreitol, and 25 µg/ml
bovine serum albumin) and treated with UDG (0.4, 4, or 40 fmol) at
37 °C for 10 min. The reaction was stopped with 5 µl of 0.2 M NaOH, heated at 90 °C for 10 min, dried in the Speed Vac (Savant), and dissolved in 10 µl of formamide dye (80%
formamide, 0.05% xylene cyanol FF and bromphenol blue, and 1 mM Na2EDTA). Aliquots (5 µl) were analyzed on
18% polyacrylamide, 8 M urea gels (21), and the bands
corresponding to substrate and product were quantitated by a Bioimage
Analyzer (Fuji). Control reactions were treated exactly in a
similar manner, except that no protein was added.
-galactosidase reporter systems
(16). The open reading frames corresponding to EcoSSB,
MtuSSB, EcoUDG, and MtuUDG were polymerase chain reaction-amplified from the respective pTrc99C based
constructs, using forward (5'-GGAATTCCACAGGAAACAGACCAT-3') and
reverse (5'-CTTTGATCATCCGCCAAAACAGCC-3') primers and Pfu DNA polymerase. The polymerase chain reaction products were digested with
EcoRI and PstI enzymes and cloned into the
corresponding sites of pGBT9 and pGAD424 vectors. The recombinants were
verified by restriction analyses and nucleotide sequencing.
1) at 4 °C. The complex and the free DNA bands
were visualized by autoradiography (21).
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain (3, 4). In this study, we assayed
the various SSB preparations from E. coli BW310
(ung
) and E. coli BL21(DE3)
(ung+) for the presence of UDG activity. As
shown in Fig. 1, EcoSSB and
MtuSSB from the ung+ E. coli show complete excision of uracil from Loop-U2 (lanes 4 and 6), whereas those from the ung
strain do not exhibit any UDG activity (lanes 3 and
5). On the other hand, when a similar assay was carried out
with EcoRRF purified from an ung+
strain of E. coli BL21 (DE3), no uracil excision was seen
(lane 7), indicating that the SSB-UDG interaction is
specific. Thus, our first indication of a possible interaction between
these two classes of proteins comes from the observation that the SSB
preparations from ung+ E. coli
contained UDG activity despite the fact that the purification schemes
for both of the proteins utilize significantly different steps (17,
24).
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Fig. 1.
Association of UDG activity with SSBs.
The 5' 32P end-labeled Loop-U2 (1 pmol) was mixed with 5 pmol of either EcoSSB (lanes 3 and 4)
or MtuSSB (lanes 5 and 6) or with
EcoRRF (lane 7) purified from
ung or ung+ strains of E. coli as indicated and assayed for UDG activity (see "Materials
and Methods"). Control reactions were carried out with buffer alone
(lane 1) or an excess (4 pmol) of EcoUDG
(lane 2). The bands marked S and
P correspond to the substrate and product,
respectively.
-galactosidase assays using S. cerevisiae SFY256 (Table I) reflect
the findings of the HIS3 reporter in that the LacZ values
obtained for the interaction between SSB and UDG proteins varied from
24 to 40 Miller units (Table I). Although these values are about
20-30% of that of the native GAL4 activator, they are significantly
higher than the values obtained for various negative controls (0.8-1.3
Miller units).
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Fig. 2.
Interaction between
MtuSSB, EcoSSB,
MtuUDG, and EcoUDG using the yeast
two-hybrid analysis. MtuSSB, EcoSSB,
MtuUDG, and EcoUDG were cloned as fusions with
the binding domain (pGBT9) or the activation domain (pGAD424) of GAL4
protein and cotransformed into the host strain (HF7c). The host strain
alone and the transformants were streaked on medium lacking histidine,
leucine, and tryptophan and containing 10 mM
3-aminotriazole. Sector 1, HF7c; sector 2,
pGBT9EcoUDG and pGAD424EcoSSB; sector
3, pGBT9EcoUDG and pGAD424
MtuSSB; sector 4, pGBT9MtuUDG and
pGAD424MtuSSB; sector 5, pGBT9EcoUDG
and pGAD424EcoUDG; sector 6,
pGBT9MtuUDG and pGAD424EcoSSB; sector
7, pGBT9EcoSSB and pGAD424EcoSSB;
sector 8, pGBT9MtuSSB and
pGAD424MtuSSB.
Interactions of various SSBs with UDGs as indicated by
-galactosidase activity
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Fig. 3.
Diagrammatic sketches of the various SSBs
used. EcoSSB and MtuSSB are similar in the
region upstream of amino acid position 129 (standard E. coli
numbering, which corresponds to position 130 in the genes for both
EcoSSB and MtuSSB). The MtuSSB gene
contains a conveniently located BamHI site at this position.
A BamHI site was introduced at position 129 of the
EcoSSB gene and used to generate chimeric constructs by
domain swapping at the site (16). EcoMtuSSB contains the N-
and C-terminal domains from EcoSSB and MtuSSB,
respectively. The MtuEcoSSB contains the N- and C-terminal
domains from MtuSSB and EcoSSB,
respectively.
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Fig. 4.
Far Western blot analyses. The profiles
of various purified proteins (SSBs and EcoRRF, ~2 µg of
each) on a 15% polyacrylamide gel containing 0.1% SDS under reducing
conditions as detected by Coomassie Blue staining
(A). The SSBs and EcoRRF (~2 µg of
each) and UDGs (~100 ng of each) (B and D) or
the SSBs and EcoRRF (~10 µg of each) (D and
E) were electrophoresed on SDS-PAGE, transferred to a
polyvinylidene difluoride membrane, incubated with EcoUDG
(B and D) or MtuUDG (C and
E), and detected with the respective anti-UDG antibodies
(see "Materials and Methods").
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Fig. 5.
Supershift assays. 5' end-labeled
single-stranded oligomer (1 pmol) was either not incubated with SSBs
(lanes 1 and 4) or incubated with 10 pmol of
EcoSSB or MtuEcoSSB without (lanes 2 and 5, respectively) or with 20 pmol of EcoUDG
(lanes 3 and 6, respectively) for 30 min and
analyzed as described under "Materials and Methods." B,
all treatments are the same as described in A, except that
MtuSSB and EcoMtuSSB were used.
7 and 1.4 × 10
7
M, respectively). Of the heterologous combinations,
MtuSSB with EcoUDG showed lower affinity
(KD, 8.4 × 10
6 M)
relative to the homologous combinations of SSBs and UDGs, whereas the
EcoSSB with MtuUDG did not show a detectable
interaction (4). Because the far Western analysis (Fig. 4E)
suggested that there was a weak interaction between EcoSSB
and MtuUDG, we reinvestigated our SPR analysis with regard
to this heterologous combination of proteins. Upon increasing the
concentrations of MtuUDG (up to 10.5 µM), a
weak interaction between these proteins could also be discerned
(KD, 7.67 × 10
6 M;
Table II).
Kinetic and equilibrium constants of SSBs and UDGs interactions
6 and 4.46 × 10
6 M, respectively) is substantially lower
than those of homologous combinations of SSBs and UDGs. However, a
striking outcome of these observations and the far Western analyses
(Fig. 4C) is that although the EcoMtuSSB
interacts with MtuUDG (KD, 2.4 × 10
6 M), it is incapable of interacting with
EcoUDG. Therefore, the interaction of EcoUDG with
EcoSSB and MtuEcoSSB but not with
EcoMtuSSB suggests that the EcoSSB contacts
EcoUDG through its C-terminal domain. Taken together, these
findings show that the C-terminal domains of both EcoSSB and
MtuSSB are primarily responsible for their interactions with
the respective UDGs, and at least in the case of EcoSSB, the
data allow us to conclude that its C-terminal domain is necessary and
sufficient to engage EcoUDG as one of its interacting
partners. On the other hand, the existence of weak interactions between
MtuUDG and MtuEcoSSB or EcoSSB (Fig. 4E and Table II) does not permit us to completely rule out
the contribution of the N-terminal domains of the SSBs (possibly
through the conserved amino acids) for the interactions involving the mycobacterial proteins.
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Fig. 6.
Effect of MtuEcoSSB and
EcoMtuSSB on uracil excision from structured
substrate, Loop-U2. A, the 5' 32P-labeled
Loop-U2 oligonucleotide (1 pmol) was either not mixed (lanes
4-6) or mixed with 5 pmol of MtuEcoSSB (lanes
7-9) or EcoMtuSSB (lanes 10-12) and
treated with EcoUDG. Lanes 1 and 2 show the control reactions with 5 pmol of MtuEcoSSB
(ME) and EcoMtuSSB (EM), and
lane 3 shows the buffer control. B, the 5'
32P-labeled Loop-U2 oligonucleotide (1 pmol) was either not
mixed (lanes 1-4) or mixed with 5 pmol of
MtuEcoSSB (lanes 5-7) or EcoMtuSSB
(lanes 8-10) and treated with MtuUDG. Lane
1 represents the buffer control. The bands marked
S and P correspond to the substrate and product,
respectively.
ssb strain of E. coli (16).
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Fig. 7.
Interaction of the chimeric SSBs with
Loop-U2. Loop-U2 (1 pmol) was incubated with different amounts of
SSBs (0.1, 0.5, 1, 2.5, 5, 7.5, 10, and 15 pmol), and the complex
formation was analyzed by EMSA. The free and complex bands were
quantitated by Bioimage Analyzer. The fraction (%) of Loop-U2 bound
(bound/(free + bound)) × 100 was plotted against the amounts of
MtuEcoSSB and EcoMtuSSB.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of the DNA polymerase, primase, RecA, and the
proteins involved in DNA repair such as UvrD, exonuclease I, MucB, and
MucA (25-30). By using the chimeric constructs in studies employing
the far Western analysis, SPR, and EMSA, we show that the C-terminal
domain of SSB is responsible for interacting with UDG. The
EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary
(DNA-SSB-UDG) complex. However, neither MtuSSB nor the
chimera containing the N-terminal domain from EcoSSB shows
such interactions. Thus, the C-terminal domain (48 amino acids) of
EcoSSB is necessary and sufficient for interaction with
EcoUDG. Similarly, the C-terminal domain of
MtuSSB mediated the interaction with MtuUDG.
However, weak interactions of MtuUDG with EcoSSB
and the chimeric SSB lacking the C-terminal domain of MtuSSB
(MtuEcoSSB) suggested that the conserved amino acids in the
N-terminal domains of SSBs might also contribute to their association.
Further, from the activity assays shown in Fig. 6, it appears that the
effect of SSBs on uracil excision from the structured oligomer,
Loop-U2, is mediated through the C-terminal domain. The crystal
structure of EcoSSB shows the C termini as flexible arms
extending out of the globular central structure (9). This extensible
structure of the C terminus supports the view that it is able to
mediate interactions with proteins in vivo.
, FEN1, and DNA ligase I at the
replication foci (14). The increasing evidence for the involvement of
replication protein A in excision repair sets a precedent for a similar
scenario in the prokaryotic systems and hints at the universality of
the phenomenon.
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ACKNOWLEDGEMENT |
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The instrument facility, which is supported by the Department of Biotechnology, is acknowledged for the use of the Bioimage Analyzer and BIAcore.
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FOOTNOTES |
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* This work was supported in part by research grants from the Council of Scientific and Industrial Research, the Department of Biotechnology, and the Department of Science and Technology, New Delhi, India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a senior research fellowship from the Council of
Scientific and Industrial Research.
§ To whom correspondence should be addressed. Tel.: 91-80-309-2686; Fax: 91-80-360-2697 or 91-80-360-0683; E-mail: varshney@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100393200
2 A. R. Rao and U. Varshney, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: UDG, uracil DNA glycosylase; SSB, single-stranded DNA binding protein; EcoSSB, E. coli SSB; EcoUDG, E. coli UDG; MtuSSB, M. tuberculosis SSB; MtuUDG, M. tuberculosis UDG; EcoRRF, E. coli ribosome recycling factor; MtuEcoSSB, contains the N-terminal portion from MtuSSB and the C-terminal domain (48 amino acids) from EcoSSB; EcoMtuSSB, contains the N-terminal domain from EcoSSB and the C-terminal domain (34 amino acids) from MtuSSB; EMSA, electrophoretic mobility supershift assay(s); SPR, surface plasmon resonance.
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