From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India
Received for publication, August 29, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Some members of the DNA-binding protein from
stationary phase cells (Dps) family of proteins have been shown to play
an important role in protecting microorganisms from oxidative or
nutritional stress. Dps homologs have been identified in various
bacteria such as Escherichia coli, Bacillus
subtilis, and Listeria innocua. Recently we have
reported the presence of a Dps homolog, Ms-Dps, in Mycobacterium
smegmatis. Ms-Dps was found to have a nonspecific DNA binding
ability. Here we have detected two stable oligomeric forms of Ms-Dps
in vitro, a trimeric and a dodecameric form. Interestingly, the conversion of Dps from a trimeric to a dodecameric form takes place
upon incubation at 37 °C for 12 h. These two oligomeric forms
differ in their DNA binding properties. The dodecameric form is capable
of DNA binding and forming large crystalline arrays with DNA, whereas
the trimeric form cannot do so. However, even in the absence of DNA
binding, the trimeric form has the capacity to protect the DNA against
Fenton's-mediated damage. The protection is afforded by the
ferroxidase activity of the trimer. However, the trimeric form cannot
protect DNA from DNaseI attack, for which a direct physical shielding
of DNA by the dodecamer is required. Thus we suggest that Ms-Dps
provides a bimodal protection of DNA by its two different oligomeric forms.
Microorganisms have developed efficient mechanisms to adapt
rapidly and to survive a variety of chemical and physical stress conditions (1). Generation of reactive oxygen species
(ROS)1 is one such stressful
condition. ROS are potent cellular oxidizing agents that damage
proteins, membrane lipids, and DNA (2-3). During aerobic growth,
generation of ROS and of hydrogen peroxide (H2O2) is unavoidable. Reaction of
H2O2 with free transition metals like ferrous
iron can result in the formation of highly reactive hydroxyl radicals
(OH·) (4). To minimize damages through such
ROS, microorganisms have evolved a number of protective ways that help
in maintaining the biomolecules in native state. ROS scavenging enzymes
such as superoxide dismutases, catalases, and peroxidases, oxidative damage repair enzymes (2, 3), and a nonspecific DNA binding and
protecting protein, Dps, (DNA binding protein from stationary phase cells) (5) are a few examples in this
category. Almost all the bacteria when exposed to ROS exhibit an
adaptive response by switching on the expression of genes coding for
these proteins (6). Such strategies are all the more important for
pathogenic bacteria because production of reactive oxygen species is a
major killing mechanism adopted by many hosts. These schemes also
become important during the growth of the organism in stationary phase
or during nutrient limiting condition. Thus, the regulation of gene
expression upon the induction of starvation and during the stationary
phase has been an area of intense research.
In the stationary phase cultures of Escherichia coli, the
existence of a novel protein Dps was discovered around a decade ago
(5). It is a nonspecific DNA binding protein with a structure very
similar to ferritins. Because of their structural homology they have
been classified under the same superfamily in the SCOP data base (7).
Despite being a DNA binding protein, Dps lacks any of the known DNA
binding motifs. Even though the crystal structure of E. coli
Dps has been solved (8), the exact mechanism with which it binds to DNA
is not fully understood. Whatever the mechanism, Dps provides a novel
way of binding to DNA. Highly crystalline and ordered assemblies of
Dps-DNA complexes have been identified both in vitro (5) and
in vivo (9).
Functionally, Dps has been shown to protect the cells against oxidative
radicals generated by Fenton's reaction and also against various other
DNA damaging agents (10, 11). Because the predominance of such radicals
becomes greater during the stationary phase of the bacterial growth
cycle, Dps expression has also been shown to be induced in stationary
phase and upon nutrient starvation (12). Besides its protective role,
Dps also has a global regulatory role in controlling gene expression
during prolonged starvation (5).
Dps-like proteins have been identified in distantly related bacteria
such as E. coli, Bacillus subtilis,
Listeria innocua, and Synechococcus sp. (5,
13-15). We have earlier reported the presence of a Dps homolog,
Ms-Dps, in Mycobacterium smegmatis (16). In this study we
throw further light on the DNA binding ability of Ms-Dps. We show that
only the dodecameric species of Ms-Dps is capable of complex formation
with DNA. We also report a bimodal type of protection of DNA by Ms-Dps,
one without physical interaction with DNA and the other by direct
binding to DNA. Last, the importance of Ms-Dps as an evolutionary link
between ferritins and Dps is also discussed.
Bacterial Strains and Plasmids--
The DH5 Ms-Dps Purification--
Ms-Dps purification was performed as
described earlier (16). In brief, E. coli strain BL21 DE3
(pLys) was transformed with pET-dps. These cells were
grown at 37 °C in Luria Bertani medium to an
A600 of 0.5 and then induced with 1 mM
isopropyl-1-thio- Native Polyacrylamide Gel Electrophoresis (PAGE)--
A 10%
native polyacrylamide gel was prepared using discontinuous buffer
system according to the method of Laemmli (19). The gel recipe was the
same, excluding the presence of sodium dodecyl sulfate (SDS). Samples
were loaded with a dye (10% glycerol, 0.002% bromphenol blue), and
the electrophoresis was carried out at a constant current of 15 mA.
Gels were then stained with Coomassie Brilliant Blue R250. Bovine serum
albumin (BSA) and horse spleen ferritin were used as markers.
Gel Retardation Assays--
pUC19 DNA was mixed with Ms-Dps at a
DNA:protein molar ratio of 1:102 or 1:103 in 50 mM Tris-HCl (pH 7.9), 50 mM NaCl. The
incubation was carried out at 30 °C for 30 min. Wherever indicated,
Ms-Dps was incubated at 37 °C for 12 h prior to the DNA
binding. The complex was then resolved on a 1% agarose gel in 0.5%
TBE buffer consisting of 89 mM Tris borate (pH 8.0) and 1 mM EDTA. The electrophoresis was carried out at a constant
voltage of 50 V. The unbound free and protein-bound DNA was then
detected by ethidium bromide staining.
Staining of Iron-binding Proteins--
Purified Ms-Dps was first
incubated at 37 °C for 6 h to allow partial oligomerization.
100 µg of this preparation was then incubated with 1 mM
ferrous sulfate in 50 mM Tris-HCl (pH 7.9), 50 mM NaCl for 1 h at room temperature. The products were
resolved on a 10% native PAGE. The gel was then stained with potassium ferricyanide solution, 100 mM K3
(Fe(CN)6) in 50 mM Tris-HCl, pH
7.5, 100 mM NaCl, for 10 min in the dark and destained with 10% trichloroacetic acid/methanol solution. After taking an image of
the stained gel, it was subjected to Coomassie Blue staining using
standard techniques. Horse spleen ferritin and BSA were used as
positive and negative controls, respectively.
In Vitro DNA Damage Assay--
pUC19 DNA was used to assess the
ability of Ms-Dps to protect DNA from oxidative damage. In a total
reaction volume of 20 µl containing 50 mM Tris-HCl (pH
7.9), 50 mM NaCl, Ms-Dps was allowed to interact with pUC19
DNA at 30 °C for 30 min. Then to assay for oxidative damage,
FeSO4 was added at a concentration of 25 or 50 µM and incubated for 5 min, followed by further addition of 5 mM H2O2 and incubation for
another 5 min. To check for DNA damage induced by DNaseI,
MgCl2 was added to a final concentration of 40 mM followed by treatment with 1 unit of DNaseI (1 unit is defined as the amount of enzyme causing an increase in absorbance at
260 nm by 0.001 per minute) for 5 min at room temperature. The
reactions were stopped with 50 mM EDTA. The products of the reactions were resolved on a 1% agarose gel in 0.5% TBE buffer and
stained with ethidium bromide. Wherever indicated, Ms-Dps was incubated
at 37 °C for 12 h prior to the DNA damage assays.
Electron Microscopic Analysis--
Ms-Dps after incubation at
37 °C for 12 h alone and the Ms-Dps-DNA complexes were
placed on copper grids. After 2 min of absorption at room temperature,
the samples were negatively stained with uranyl acetate for 5 min.
Specimens were examined in a Jeol 100 CxII electron microscope at 80 kV. The photographs were taken at ×65,000 magnification. The diameters
of the rings were measured from electron microscopic negatives with the
aid of a Wild-Heerbrugg MPS12 zoom stereomicroscope. About 40 numbers
of differentially placed rings were measured.
Spectroscopic Analysis of Iron Incorporation--
The iron
oxidation and incorporation kinetics were followed
spectrophotometrically at 305 nm on a Jasco V-530 spectrophotometer. The solution of Ms-Dps (6 µM) was initially scanned for
300 s; subsequently, freshly prepared 10 µM ferrous
sulfate was added and scanned again for 300 s. As a control the
rate of Fe2+ auto-oxidation was measured in parallel.
Temperature-induced Change in the Oligomeric Status of
Ms-Dps--
We have recently reported the identification,
purification, and DNA binding ability of Ms-Dps (16). However, in the
gel retardation assay, even at a large excess of Ms-Dps over DNA, generation of higher molecular weight species was not observed, unlike
in the E. coli DNA-Dps complex (5). Because all the Dps
family members are known to form multimers and different oligomeric forms might have different DNA binding abilities, it was thought that
the oligomeric status of Ms-Dps should first be identified. For this
purpose, protein was subjected to native PAGE analysis. The recombinant
protein when purified under native conditions shows the presence of two
major species on a 10% native PAGE (Fig. 1, lanes 1-3), one lower
oligomeric form that runs near BSA (MW-63,000) and another
higher oligomeric form that runs near ferritin (MW-450,000). Both BSA
and ferritin are globular, acidic proteins with isoelectric points
similar to that of Ms-Dps (BSA-4.8, ferritin-4.5, Ms-Dps-5.4). Thus
they can be used as markers in native PAGE analysis. The mobility of
the lower oligomer on the gel indicates that it could be a trimeric
species (MW of Ms-Dps monomer-216,000). As is evident in Fig. 1,
lane 3, the trimeric form is predominant over the higher oligomeric form. This preparation of protein was then checked for its
DNA binding ability. As shown in Fig. 2,
the protein did not retard the pUC19 DNA on a 1% agarose gel even at
1:103 DNA: protein molar ratio.
When this protein preparation, the purification of which had been
carried out at 4 °C, was incubated at 37 °C for 12 h and checked on 10% native PAGE, the higher oligomeric form became predominant over the trimeric form (Fig. 1, lane 4). The
switch in relative ratio of trimer to higher oligomer upon 37 °C
incubation was consistently observed with different protein
preparations. The formation of the higher oligomer was found to be an
irreversible process, i.e. when incubated back at 4 °C or
at room temperature the higher oligomer did not dissociate into the
trimer. It is possible that the formation of the higher oligomer is an
energy-requiring process. The probability of temperature-induced
structural changes in the monomers, which favor higher oligomer
formation, also cannot be ruled out. However, the secondary structures
of Ms-Dps both at 4 °C and after incubation at 37 °C were the
same, as was seen by circular dichroism studies (data not shown).
A gel retardation assay was then performed after the incubation of
Ms-Dps at 37 °C for 12 h. As shown in Fig.
3, this treatment enabled the protein to
form a complex with pUC19 DNA, and the complex did not enter 1%
agarose gel. This property is similar to that observed with E. coli Dps (5, 10). It should be mentioned here that some batches of
the purified Ms-Dps showed only a slight mobility shift of DNA (16),
but upon incubation at 37 °C, the high molecular weight complex with
DNA forms instantaneously. Upon correlating the native PAGE
analysis with the gel retardation assays, it is apparent that the
presence of the higher oligomeric form of the protein is mandatory for
complex formation with DNA.
Complex Network Formation of Ms-Dps with DNA--
Electron
microscopic studies were then performed on the 37 °C-incubated
Ms-Dps and Ms-Dps-DNA complexes. Preparations of the protein alone when
visualized under electron microscope showed discrete ring-like
structures of ~9 nm diameter (Fig.
4a). This size correlates well
with the diameter of the modeled Ms-Dps dodecameric molecule, which is
8.8 nm (16) and also with that of the crystal structure of E. coli Dps, which is 9 nm (8). When the same protein was incubated
with DNA and then visualized in the electron microscope, large and
highly ordered two-dimensional arrays of the rings were seen (Fig.
4b). This honeycomb-like arrangement is very similar to that
observed with E. coli Dps-DNA complexes (5).
Electron microscopic analysis suggests that the higher oligomer seen on
the native gels is a dodecamer. Various different multimeric forms of
E. coli Dps were detected earlier (8), but it is not known
which one of these is the actual DNA binding species. Our results
presented here indicate that it is the dodecameric form of Ms-Dps,
which is capable of forming high molecular weight complexes with DNA.
Other lower oligomeric forms and the monomers do not have this ability.
Iron-binding Ability of Ms-Dps--
Some Dps family proteins, like
Dps of E. coli, the ferritin of L. innocua, and
Dpr from Streptococcus mutans have been shown to bind iron
(5, 14, 20). Therefore, in this study the iron-binding ability of
Ms-Dps was examined. First the oligomerization was allowed to proceed
by incubating purified Ms-Dps at 37 °C. The incubation was carried
out only for 6 h so as to attain a population of both the
oligomeric forms in the reaction mixture. Both oligomeric forms were
then allowed to incorporate iron by incubating with 1 mM
ferrous sulfate. The two forms were then separated on a native PAGE.
Upon staining with K3(Fe(CN)6, as is seen in
Fig. 5A, the higher oligomeric
form was stained, along with ferritin which was used as a positive
control. However, no band was visible that corresponded to the lower
oligomer. The same gel when stained with Coomassie Blue showed the
presence of the lower oligomer as well as of BSA (Fig.
5B).
The following is a structurally interesting observation. The
dodecameric structure of modeled Ms-Dps (16) has a hollow core in the
center in which ferrous ions can be incorporated. The formation of a
trimer, on the other hand, would give rise to an open structure with no
such hollow core (Fig. 6). The
trimeric species, therefore, offers no place for iron
incorporation to occur. The structures of the trimeric and dodecameric
forms of E. coli Dps as adopted from Ref. 8 are shown in
Fig. 6.
Recently it has been shown that in E. coli Dps, iron
oxidation and hydrolysis can lead to incorporation of about 500 ferric ions inside the dodecameric protein shell (21). Our experiments here
suggest that the dodecameric Ms-Dps is also capable of incorporating iron ions inside its protein shell, whereas the probable trimeric species cannot accumulate iron because of structural constraints.
Functional Aspects of Ms-Dps--
One important function of Dps
in vivo is to protect the DNA from oxidative radicals. To
investigate whether Ms-Dps also has this protective ability, an
in vitro DNA damage assay was performed. H2O2 in the presence of ferrous ions generates
OH· radicals through the Fenton reaction:
Fe2+ + H2O2
The OH· radicals thus generated have a
DNA-nicking ability. Various concentrations of ferrous ions and
H2O2 were used to bring about DNA damage. As
seen in Fig. 7, in the presence of 25 µM FeSO4 and 5 mM
H2O2 all the supercoiled pUC19 DNA was nicked, resulting in relaxed DNA, whereas in the presence of Ms-Dps, DNA was
protected against this nicking. At 50 µM ferrous sulfate
and 5 mM H2O2 the DNA was totally
degraded, whereas it remained intact in the presence of Ms-Dps. This
experiment shows that Ms-Dps has the ability to protect DNA against
Fenton's-mediated damage.
The protein used in this assay had not undergone 37 °C incubation.
Because trimeric Ms-Dps does not bind DNA, it was quite interesting to
note its DNA protection ability from the free radical onslaught. As
expected, Ms-Dps after incubation at 37 °C also showed DNA
protection in the same assay in the presence of FeSO4 and
H2O2 (Fig.
8).
These observations point toward an important characteristic of the
protein. Ms-Dps seems to provide a bimodal type of protection to DNA.
When the protein is predominantly in trimeric form, it does not bind
DNA. Even in the absence of direct physical interaction with the DNA,
the trimeric form is capable of protecting the DNA from
Fenton's-mediated damage. Under the conditions where it forms the
dodecamer, it generates a complex network with the DNA and thus
protects it from damage. This type of protection might also be
important against various other DNA-damaging agents such as nucleases,
alkylating agents, and chemical mutagens. The capability of these two
oligomeric forms to protect DNA against DNaseI was then tested. As
shown in Fig. 9, DNA incubated with the
trimeric form of Ms-Dps was totally degraded by DNaseI, whereas upon
incubation with the dodecameric form, DNA remained intact. It can be
inferred from this experiment that because the trimeric form does not
bind DNA, DNA was free to be digested by the endonuclease DNaseI.
Direct physical interaction of Ms-Dps with DNA is required to protect it from an enzyme like DNaseI, which can be afforded only by the dodecameric form. Thus the mechanism of protection provided by the two
oligomeric forms of the same protein is not the same. The higher
oligomer protects by physically shielding the DNA, whereas the lower
oligomer protects without even directly interacting with DNA.
The latter mode of protection can be explained mechanistically if the
protein has an iron-chelating activity. Although we have not seen any
iron incorporation ability in the trimeric species (Fig. 5), it does
not rule out the possibility of the trimer having iron-binding and
ferroxidase activity. As has been reported recently (22), E. coli Dps has a ferroxidase activity, i.e. conversion of
Fe2+ to Fe3+ using H2O2
as an oxidant. The protein has been shown to form coordinate complexes
with Fe2+, which is then oxidized to
Fe3+ using H2O2 via a mechanism
that does not allow Fenton's reaction to proceed. The generation of
OH· radicals is thereby inhibited. We thus
speculated that the lower oligomeric form of Ms-Dps could also be
protecting the DNA through such a ferroxidase activity. To check this
hypothesis spectral analysis at 305 nm was employed, because
Fe3+ species absorbs at 305 nm where Fe2+ does
not. As shown in Fig. 10A,
the buffer alone did not absorb at 305 nm. However, upon addition of
FeSO4, the absorbance gradually increased with time, which
implies that Fe2+ was converted to Fe3+ by
utilizing molecular oxygen of air. Because the spectrum was taken only
until 300 s, all of the ferrous ions did not get oxidized to
ferric ions in this short span of time, and thus the saturation was not
reached. In the presence of protein, when FeSO4 was added there was a sudden exponential increase in the absorbance, which later
reached saturation (Fig. 10B). The presence of protein
allowed the conversion of total ferrous ions to ferric ions within
300 s. This clearly shows that the trimeric Ms-Dps has a
ferroxidase center that can rapidly oxidize Fe2+ to
Fe3+ using molecular oxygen of air. Addition of
H2O2 after 300 s did not bring about any
further change in the absorbance (data not shown).
We would like to speculate here that the trimeric form of Ms-Dps is
capable of protecting DNA without physically interacting with it,
because of its ability to chelate out Fe2+ ions in the
vicinity of DNA and then oxidize them to Fe3+ using
molecular oxygen. This observation is in contrast to that of a recent
report (22), in which the authors found that the E. coli Dps
cannot utilize oxygen to convert Fe2+ to Fe3+
effectively. H2O2 was shown to be a better
oxidant than oxygen. However, this property is unlike those of
ferritins, because ferritins utilize oxygen as an oxidant. Therefore it
appears that Ms-Dps is exhibiting ferroxidase property similar to that
of ferritins and not like that of E. coli Dps. This protein
is thus a unique member of the Dps family with a DNA binding ability
like that of Dps and a ferroxidase activity like that of ferritins. One of the members of the family, Dps of L. innocua, has been
identified as a true dodecameric ferritin functioning in iron storage
(14), but this protein does not have DNA binding ability. Recently Dlp1 and Dlp2, the two Dps-like proteins from B. anthracis, have
been designated as mini-ferritins (23) because they are ferritins that
are dodecamers rather than the usual 24-mers. They also do not bind
DNA. Although Dps A of Synechococcus sp. is a DNA-binding hemoprotein possessing a weak catalase activity, it is not known to
have a ferroxidase activity (15). To date, no other single member of
this family is known to possess both DNA-binding and ferritin-like
ferroxidase activities. As had been discussed elsewhere (15), Dps
proteins might have evolved as metal-binding proteins that later
acquired DNA binding ability. The Ms-Dps, thus, could be a link between
the two extremes, having a DNA binding property while still retaining
the ferroxidase activity of ferritins.
During the evolution of life, the appearance of atmospheric oxygen
offered the opportunity to utilize molecular oxygen as the oxidant in
respiration. This provided energetic advantages over fermentation and
respiratory pathways, which rely on other oxidants. However, the
presence of intracellular oxygen also allowed unavoidable production of
reactive oxygen species, which damage critical biomolecules. In most
cases toxicity is exerted because of their direct damaging effects on
DNA. A number of preventive mechanisms evolved since then to take care
of such ROS-mediated toxicity.
One important question that has been quite frequently addressed is the
protection of DNA from ROS in a bacterial cell under stationary phase
or under some kind of nutritional stress. Because of constraints of
resources under these conditions, many energetic expensive mechanisms
of DNA protection cannot be employed. This question attains further
importance in mycobacterial species because the latent pathogenic
mycobacteria can survive within the host for a very long time devoid of
all necessary nutrients and later can resume growth at an appropriate
moment (24). Naturally, the organism should be able to adopt efficient
mechanisms to protect its genetic material under such nutritionally
stressful conditions. Recently we have analyzed the proteome of
M. smegmatis under carbon starvation with the aim of
identifying some such mechanisms (16). Although M. smegmatis
is a non-pathogenic species, we thought it would be worthwhile to
identify the proteins that are overexpressed under carbon starvation,
because then attempts could be made to correlate the expression of such
proteins in its pathogenic counterpart. Comparatively faster growth
kinetics of M. smegmatis under carbon starvation (25) and
the known genome sequence of Mycobacterium tuberculosis (26)
help in such an endeavor. Through such an exercise we have identified a
Dps-like protein in M. smegmatis (16).
Although the role of Dps has been worked out in E. coli and some other organisms as mentioned earlier, its presence
was not reported in mycobacteria before. However, the most interesting aspect was the absence of a sequence homolog of Dps in the M. tuberculosis genome, naturally raising questions regarding its ubiquitous function in protecting DNA under starvation conditions. As
was mentioned earlier, ferritins and Dps are members of the same
superfamily of proteins and are known to have evolved as divergent
homologs from a common ancestor (15). However, the functional
complementation of these proteins is not reported in literature. Still
it is tempting to speculate here that one of the ferritins of M. tuberculosis might be performing the function performed by
Dps-like proteins in other mycobacteria, such as M. smegmatis and Mycobacterium avium.
Our results presented in this manuscript suggest that Ms-Dps has a
bimodal way of protecting DNA under free radical onslaught. Dps has
earlier been shown to cause considerable compaction of DNA upon binding
(8). Such a nonspecific complex network formation with DNA can make the
DNA inaccessible to polymerases and other important DNA-modifying
enzymes during the growth of the bacteria. At the same time, protection
of DNA against oxidative stress also has to be taken care of. Our
findings here show that Ms-Dps has a unique capability of protecting
the DNA from such oxidative damage without physically interacting with
it. This protection appears to be carried out by the ability of Ms-Dps
to chelate out Fe2+ ions in the vicinity of DNA and to
oxidize them to Fe3+ using molecular oxygen. This reaction
bypasses the Fenton's reaction by which DNA-damaging
OH· radicals are generated. Thus by oxidizing
Fe2+ to Fe3+, Ms-Dps is capable of protecting
the most important molecule of the bacterial cell in critical situations.
The temperature incubation step involved in the conversion of Ms-Dps
from non-DNA binding to DNA binding form is interesting. This indicates
that the trimeric species, the non-DNA binding form of Ms-Dps, is more
stable and naturally occurring in M. smegmatis. It needs to
take care of only the oxidative radicals generated during normal
growth. Under stress conditions such as entry into stationary phase or
nutrient deprivation, where other DNA damaging agents also come into
the picture, oligomerization of Dps into a dodecamer becomes a
necessity to provide more efficient DNA protection by physically
shielding the DNA. It should be mentioned here that for protein
purification we have grown the cells at 37 °C, and then all the
further purification steps were performed at 4 °C. The trimeric form
of Ms-Dps thus obtained could be irreversibly converted to dodecameric
form upon incubation at 37 °C, and this is intriguing. We have no
simple answer to this apparent discrepancy. It appears that in
vivo Ms-Dps maintains equilibrium between trimer and dodecamer as
a function of growth or by the participation of other factors. Perhaps
this conversion is not irreversible as we have noticed in
vitro; we are currently pursuing a detailed analysis of the same.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain of
E. coli was used for cloning purposes and the BL21 DE3
(pLys) strain was used for protein purification. pET-dps is
a pET21b derivative containing the M. smegmatis dps gene
(16). Plasmid pUC19 (17) was used for in vitro DNA
binding and DNA damage assays.
-D-galactopyranoside. Single-step purification was performed using the Qiagen Ni-NTA affinity
matrix according to the manufacturer's instructions. After checking
the purity of the protein on a 12% SDS-polyacrylamide gel, protein was
dialyzed against 50 mM Tris-HCl (pH 7.9), 50 mM
NaCl overnight and used for further analysis. Protein concentration was
determined by the method of Lowry et al. (18). For the
formation of the higher oligomer, protein at a concentration of 1 mg/ml was incubated at 37 °C for 12 h in 50 mM Tris-HCl
(pH 7.9), 50 mM NaCl.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Temperature-induced change in oligomeric
status of Ms-Dps. BSA (MW 63, pI 4.8), lane 1; horse
spleen ferritin (MW 450, pI 4.5), lane 2; Ms-Dps after
purification at 4 °C, lane 3; Ms-Dps after
37 °C-incubation for 12 h, lane 4. Species
I, probable monomer. Species II, lower oligomer.
Species III, higher oligomer.
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Fig. 2.
Absence of binding of lower oligomeric form
of Ms-Dps to DNA. Free pUC19 DNA (lane 1). Incubated
with Ms-Dps at 30 °C for 30 min in 50 mM Tris-HCl (pH
7.9), 50 mM NaCl at DNA:protein molar ratio of
1:102 (lane 2) and 1:103 (lane
3).
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Fig. 3.
Binding of higher oligomeric form of Ms-Dps
to DNA. Ms-Dps in 50 mM Tris-HCl (pH 7.9), 50 mM NaCl at 1 mg/ml concentration was incubated at 37 °C
for 12 h prior to DNA binding assay. Free pUC19 DNA (lane
1). Ms-Dps DNA complex at 1:103 DNA: protein molar
ratio (lane 2).
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Fig. 4.
Electron microscopic analysis of Ms-Dps and
Ms-Dps-DNA complexes. a and b are at the same
magnification. a, Ms-Dps alone. b, Ms-Dps-DNA
complex.
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Fig. 5.
Iron binding ability of Ms-Dps. BSA
(lane 1), horse spleen ferritin (lane 2), and
MS-Dps (lane 3) were resolved on a 10% native PAGE and
stained with potassium ferricyanide (A) and then with
Coomassie Brilliant Blue R250 (B).
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Fig. 6.
Structural comparison between E. coli Dps dodecamer and trimer (adopted from Ref.
8). A, dodecameric molecule showing the hollow
core. B, open trimeric structure.
Fe3+ + OH
+ OH·.
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Fig. 7.
The protection of DNA from
H2O2-mediated damage by trimeric Ms-Dps.
pUC19 DNA alone (lane 1): the lower band is the
supercoiled form and the upper band is the relaxed DNA.
pUC19 DNA treated with 25 µM FeSO4
(lane 2) or 50 µM FeSO4
(lane 4) for 5 min followed by 5 mM
H2O2 for 5 min. pUC19 DNA incubated with the
trimeric form of Ms-Dps (DNA: protein molar ratio 1:103)
before treatment with 25 µM FeSO4 (lane
3) or 50 µM FeSO4 (lane 5)
for 5 min followed by 5 mM H2O2 for
5 min.
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Fig. 8.
The protection of DNA from
H2O2-mediated damage by dodecameric
Ms-Dps. pUC19 DNA alone (lane 1), treated with 50 µM FeSO4 for 5 min followed by 5 mM H2O2 for 5 min (lane
2), pUC19 DNA first incubated with dodecamer at 30 °C for 30 min in 50 mM Tris-HCl (pH 7.9), 50 mM NaCl
(DNA:protein molar ratio 1:103) followed by 50 µM FeSO4 for 5 min and 5 mM
H2O2 for 5 min (lane 3). The complex
of DNA and Ms-Dps can be seen at the well.
View larger version (14K):
[in a new window]
Fig. 9.
The digestion of DNA by DNaseI in the
presence of lower and higher oligomers of Ms-Dps. pUC19 DNA
untreated (lane 1) and treated with 1U DNaseI for 5 min
(lane 2). Prior to the DNaseI treatment, DNA was either
incubated with the dodecameric form (lane 3) or the trimeric
form (lane 4) of Ms-Dps.
View larger version (11K):
[in a new window]
Fig. 10.
Ferroxidase activity of Ms-Dps.
A, absorbance of buffer alone (solid line) and
after addition of 10 µM FeSO4 (dashed
line). B, absorbance of 6 µM trimeric
Ms-Dps alone (solid line) and after addition of 10 µM FeSO4 (dashed
line).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. S. S. Indy, IISc for help in electron microscopic analysis.
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FOOTNOTES |
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* This work was supported by the Department of Biotechnology of the government of 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.
Recipient of Council of Scientific and Industrial Research fellowship.
§ To whom correspondence should be addressed. Tel.: 91-80-3942836; Fax: 91-80-3600535; E-mail: dipankar@mbu.iisc.ernet.in.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M208825200
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; Dps, DNA-binding protein from stationary phase cells; BSA, bovine serum albumin.
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REFERENCES |
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