From the Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri-
midine (THP(A) or hydroxyectoine) and
2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine)
are now recognized as ubiquitous bacterial osmoprotectants. To evaluate
the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA
interaction and on restriction-modification systems, we have examined
their effect on the cleavage of plasmid DNA by 10 type II restriction
endonucleases. THP(A) completely arrested the cleavage of plasmid and
bacteriophage Two tetrahydropyrimidine derivatives identified in
Streptomyces bacteria (1-3), one a previously unknown
metabolite, THP(A),1 and the
other previously identified (as ectoine) in halophilic bacteria (4),
THP(B), are now recognized as widely spread osmoprotectants within the
bacterial world (5). The role and activities of THPs are of special
interest as they represent a limited group of osmoprotectants that are
synthesized de novo, in the bacterial cell, in contrast to
those transported from the medium (6). Their synthesis in a number of
Streptomyces strains as a response to increased salinity and
elevated temperature was recently described (7). THPs are small
molecules, highly soluble in water and neutral at physiological pH. NMR
and x-ray crystallography data show that THPs are zwitterionic
molecules with a delocalized More information has been accumulated lately on THPs activity in living
cells. It was found that exogenously provided ectoine (THP(B)) could
reverse growth inhibition, caused by osmotic stress, in
Escherichia coli (9), Corynebacterium glutamicum
(10), and the soil bacterium Rhizobium meliloti (11). We
demonstrated that exogenously provided THP(A), like THP(B), reversed
inhibition of E. coli growth by osmotic stress, and
moreover, both THP(A) and THP(B) could stimulate growth of E. coli at an elevated temperature (43 °C) (7). Recently cloned
genes for ectoine synthesis from Halomonas elongata (12) and
from Marinococcus halophilus (13) were demonstrated to be
both necessary for halotolerance of ectoine-producing bacteria and
osmotically regulated.
The ability of osmoprotectants (e.g. proline and
betaine) to overcome the inhibitory effect of osmotic stress in
bacteria was traditionally explained in two ways. One hypothesis states that proline and betaine have special interactions with proteins that
protect them from denaturation in the presence of high concentrations of electrolytes (14, 15). In support of this mechanism, it was recently
shown that THPs stabilize proteins upon freezing and at elevated
temperatures (16, 17). According to another hypothesis, proline and
betaine are merely aimed at maintaining cell turgor in media of high
osmolarity, being compatible with normal cellular functions at high
intracellular concentration. Intracellular concentrations of THPs
attain up to 158 mM in nonhalophylic Streptomyces bacteria (7), and as high as 2.25 M
in the halophilic bacterium H. elongata (18), whereas
betaine can be synthesized to 0.6 M in the
Methanohalophilus strain Z7401 (19) and proline to 0.7 M in Bacillus subtilis (20). At these high
concentrations, proline, betaine, and THP(B) demonstrate a pronounced
destabilizing effect on DNA in vitro
(22-24).2 Moreover, THP(B)
at certain concentrations, can, like betaine (22), eliminate base pair
composition dependence of DNA melting.2 In addition, THP
molecules share similarity in structure and geometry with pyrimidine
bases and have the zwitterionic character, which was recently shown to
be responsible for modification of DNA electrostatic interaction with
counterions by a number of osmolytes (25). These notions led us to
suggest that the impact of osmoprotectants, and THPs in particular, on
protein-DNA interaction is, likely, underestimated. The destabilization
of the DNA duplex could play a dual role in the interaction of DNA with
proteins; it may either enhance binding, as it was shown for proline
and SSB protein, or diminish it, as it was shown for DNaseI, for which double-stranded DNA serves as a substrate (24). Yet the impact of
osmolytes on the sequence-specific interaction of proteins with DNA
(e.g. restriction endonucleases and transcription factors) could be even more complex and remains to be elucidated.
Type II restriction enzymes are recognized as an ideal model system for
evaluating site-specific protein-DNA interaction, as any interference
with the precise alignment of the enzyme and substrate will affect the
cleavage. It has been demonstrated that the binding of ligands, such as
antibiotics and dyes, reduces the activity and specificity of
restriction endonucleases (26). It has also been shown that the
constituents of bacterial cells, such as polyamines and basic proteins
(27) or polyphosphate (28), are also capable of inhibiting the DNA
cleavage by restriction endonucleases in vitro. Hence, the
primary function of the restriction-modification system, aimed at
protecting bacteria from phage infection or from other sources of
foreign DNA, might be dependent on the composition of intracellular metabolites.
In the present study, we have investigated the effect of bacterial
osmolytes, such as THP(A), THP(B), proline, and glycine betaine, on DNA
cleavage by several type II restriction endonucleases, taken as a model
system for specific protein-DNA interaction. The results were compared
with the effect on proteins that bind nucleic acid nonspecifically,
such as DNaseI, RNase A, and Taq DNA polymerase. We report
here that THP(A) and, to a lesser extent, THP(B) are capable of
completely arresting the DNA cleavage by a number of restriction
endonucleases, whereas proline and glycine betaine are almost
ineffective. The effect of THPs on EcoRI binding to the
oligonucleotide, containing its recognition site, and on linear
diffusion was also examined. A possible role of THPs in the modulation
of the restriction-modification system and in gene expression under
stress is suggested.
Plasmids pGEM1 and pBR322 were isolated from E. coli
strain DH5 by alkaline lysis and purified by equilibrium centrifugation in a gradient of cesium chloride (29). Restriction endonucleases EcoRI, PvuII, AvaI, and
DraI were purchased from New England Biolabs, SspI and SmaI were from MBI Fermentas, and
SgrAI and EcoRV were from Boehringer Mannheim.
Pancreatic bovine deoxyribonuclease I, ribonuclease A, yeast RNA,
betaine monohydrate (glycine betaine), and spermine tetrahydrochloride
were from Sigma, L-proline (99.5%) was from Fluka, and
Thermus aquaticus DNA polymerase (recombinant) was from MBI Fermentas.
Preparation of Tetrahydropyrimidine Derivatives--
THPs were
prepared and purified as described previously (7). The activity of THPs
preparations in the restriction assay was checked before and after
passing through a Chelex column, and no difference was observed. THPs
preparations were further analyzed by atomic flame photometry, and no
detectable amounts of Mg, Ca, and Fe ions and less then 0.1% sodium
and potassium ions were found. The micromolar final concentration of
Na+ or K+ upon addition of THPs to the reaction
mixture is negligible in comparison to 50-100 mM potassium
in the buffer. For the preparation of 2-methyl, 4-methylcarboxylate,
5-hydroxy-3,4,5,6-tetrahydropyrimidine (methyl-THP(A)) and 2-methyl,
4-methylcarboxylate-3,4,5,6-tetrahydropyrimidine (methyl-THP(B)) (Fig.
1), an excess of thionylchloride was
gradually added to a 0.5 M solution of THP(A) or THP(B) in
methanol at 0 °C under continuous stirring. Resulting solutions were
evaporated under reduced pressure and the residue, containing
CH3-THP(A)·HCl or CH3-THP(B)·HCl, was
dissolved in water and neutralized by NaOH to pH 7.0. Water was
evaporated under reduced pressure, and the solid material was
resuspended in methanol, separated from NaCl by filtration, and dried.
The purity of methyl-THP(A) and methyl-THP(B) was determined by
1H and 13C NMR (data not shown).
Preparation of the Labeled
Oligonucleotide--
(d(CGCGAATTCGCG))2 was prepared by
solid phase synthesis (Chemical Services, Weizmann Institute of
Science), purified on a denaturing polyacrylamide gel containing 7 M urea, and desalted on Sephadex G-25. The oligonucleotide
was labeled with [ Cleavage by Restriction Endonucleases--
Cleavage of DNA by
each restriction enzyme was carried out in a total volume of 20 µl of
the reaction buffer recommended by manufacturer. The cleavage reaction
was initiated by addition of the enzyme to the incubation mixture
containing THPs or other additives and terminated by addition of EDTA
to a final concentration of 20 mM. Reaction products were
analyzed by electrophoresis on a horizontal 1% agarose gel at 3 V/cm.
Gels were stained with ethidium bromide and photographed, and pictures
were quantified by densitometry.
Cleavage of dodecadeoxynucleotide by EcoRI was carried out
in 20 µl of buffer containing 50 mM NaCl, 20 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 0.1 mM dithiothreitol, 10 mM MgCl2, and
50 mg/ml bovine serum albumin. Products of the 32P-labeled
dodecadeoxynucleotide cleavage were analyzed by electrophoresis on a
20% polyacrylamide gel containing 7.0 M urea, visualized by autoradiography and quantified by densitometry.
DNase I, RNase A, and Taq Polymerase Assays--
The DNase I
assay was performed at 37 °C in 100 µl of buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,
and 100 mM NaCl. A minimal amount of enzyme (0.3 ng/ml)
needed for complete transformation of supercoiled plasmid DNA into
nicked circular within 1 h (as determined in a separate experiment
in the absence of THPs) was added to the reaction mixture containing
THPs. Aliquots, taken after short intervals of incubation (every 5 or
10 min), were immediately transferred into liquid nitrogen and kept
frozen until loaded on the agarose gel.
RNase A assay was carried out at 37 °C in 20 µl of buffer
containing 1 µg of yeast RNA, 20 mM Tris-HCl, pH 7.2, 50 mM NaCl, and 2 mM MgCl2. The
minimal amount of enzyme needed for 50% digest of RNA within 30 min
(0.2 µg/ml, as determined in a separate experiment in the absence of
THPs) was added to the reaction mixture containing THPs. Reaction
products were analyzed by electrophoresis on a 1% agarose gel.
Taq DNA polymerase activity assay was performed at 72 °C
in a 15-µl reaction mixture containing 67 mM Tris-HCl (pH
8.8); 6.7 mM MgCl2; 50 mM NaCl; 1 mM Gel-shift Assay--
Assays on EcoRI binding to the
dodecadeoxynucleotide were carried out in 20 µl of binding buffer
containing 50 mM NaCl, 20 mM Tris-HCl (pH 7.4),
0.1 mM EDTA, 0.1 mM dithiothreitol, and 50 mg/ml bovine serum albumin. THPs were added to the reaction mixture and
incubated at room temperature for 15 min before or after the addition
of EcoRI, 8 µl of the loading buffer (40% w/v sucrose and
100 µg/ml bromphenol blue) was added, and the samples were loaded on
the 9% (29:1, acrylamide:bisacrylamide) polyacrylamide gel, 45 mM Tris borate (pH 8.0), and 2.0 mM EDTA. Gels
were prerun for 2-3 h prior to loading of samples and after
electrophoresis for 3-4 h at 10-12 V/cm and 4 °C, dried, and
autoradiographed, and the resulting negatives were subjected to densitometry.
Effect of THPs on the Linear Diffusion of Restriction
Endonucleases--
We have basically followed the experiment
developed by Ehbrecht et al. (31). To evaluate the
contribution of linear diffusion to the rate of the
EcoRI-catalyzed reaction, the difference in the cleavage
rate of short (378 bp) and long (4361 bp) fragments of pBR322 plasmid
DNA with a centrally located target site by EcoRI
endonuclease (see Fig. 5) was measured. To prepare short and long DNA
fragments, 300 µg of pBR322 plasmid DNA was cleaved overnight by
SspI/EcoRV or PvuII endonucleases (60 units of each) in the buffers recommended by the manufacturers. The
resulting digest mixture was used without further purification as a
substrate for EcoRI restriction endonuclease. As was shown
previously, the components of the primary reaction do not affect the
secondary reaction (31). This experimental setup ensures that factors of the reaction other than the length of the substrate are invariant; namely, all fragments with the EcoRI site have identical
flanking sequences, and due to the presence of fragments not containing the EcoRI site, nonspecific binding is identical in all
experiments. The secondary reaction (with EcoRI
endonuclease) was carried out in the optimized buffer (31) containing
20 mM Tris-HCl, pH 7.2, 0.05 mg/ml bovine serum albumin, 50 mM NaCl, and 1 mM MgCl2 in the
presence of varying amounts of THPs.
Effect of THPs and Osmolytes on DNA Cleavage by EcoRI
Endonuclease--
We have studied the effect of THP(A), THP(B),
proline, and betaine on plasmid DNA cleavage by EcoRI
endonuclease. The rate of cleavage of pBR322 DNA by EcoRI
endonuclease was notably decreased, starting from 0.1 mM
THP(A). It is worth noting that no intermediate products such as open
circular DNA were accumulated upon inhibition (Fig.
2A). To determine the type of
inhibition and estimate the inhibition constant, the steady state
kinetics of cleavage was measured at constant DNA concentrations and
varying concentrations of THP(A). A Dixon plot (32) of these data (Fig.
2B) with Vmax constant within the
range of error, combined with a set of parallel lines in a
Cornish-Bowden plot (33) (Fig. 2C), indicates a competitive inhibition (34) with Ki = 0.16 ± 0.04 mM. The inhibition effect of other osmolytes was much less
pronounced; i.e. whereas THP(A) completely arrested plasmid
DNA cleavage at around 1.0 mM, THP(B) was 10-fold less
effective, betaine and proline at low concentrations showed stimulation
of DNA cleavage, and a notable inhibition was observed only at 100 mM (Fig. 3). It is worth
noting that at this high concentration, additional effects, such as a decrease of water activity, a significant increase of the medium dielectric constant, or changes in the pH of the reaction mixture, are
possible. The inhibitory effect of THP(A) on the EcoRI
endonuclease reaction was not unique for the pBR322 plasmid DNA as a
substrate; the same effect was observed for pGEM1 plasmid DNA and for
bacteriophage
The cleavage of dodecadeoxynucleotide duplex by EcoRI was
tested in the presence of THP(A) with the minimal amount of enzyme needed for complete cleavage and with a 5-fold excess of the enzyme. Similar results were observed in both cases (Fig.
4, A and B). The
complete (95-100%) inhibition of the reaction was reached at 5 mM THP(A). The higher concentration of THP(A) needed for complete inhibition of the oligonucleotide cleavage (3-5-fold) as
compared with plasmid DNA could be attributed to the different reaction
conditions employed in the two experiments, different flanking sequence
of recognition sites, and known differences in the reaction kinetics of
restriction enzymes on short duplexes of 8-12 bp compared with those
on longer DNA (35).
The inhibition of EcoRI endonuclease reaction by THP(A) was
not compensated by increased concentration of MgCl2 (20 mM instead of 10 mM) in the reaction mixture,
suggesting that sequestering of Mg2+ ions is not
responsible for the observed effect. In both cases, the inhibition of
DNA cleavage by THP(A) was similar to that depicted in Fig. 3.
Do THPs Affect the Linear Diffusion of Endonuclease along the
DNA?--
The cleavage of the plasmid DNA substrate by restriction
endonucleases is preceded by a number of consecutive events, such as a
nonspecific association with the DNA and linear diffusion of the
nonspecifically bound protein along the DNA until the recognition site
is reached and specific binding occurs. On exploring the possible
targets for THP(A) action, we further investigated its effect on the
linear diffusion of EcoRI along the DNA by using the
experimental approach developed by Ehbrecht et al. (31). To
ensure that the difference in the rate of cleavage in this experiment
originates solely from the chain length of the substrate, the plasmid
DNA (bearing a single EcoRI site) was cleaved beforehand by
other restriction endonucleases, producing two sets of DNA fragments of
different length. The resulting mixture was used as a substrate for
EcoRI endonuclease, thus excluding possible effect of
different flanking sequences and equalizing the nonspecific binding.
Plasmid pBR322 was cleaved by PvuII endonuclease, to yield a
linear fragment of 4361 bp. Alternatively, plasmid DNA was cleaved by
EcoRV/SspI, resulting in a 3983-bp fragment
without a recognition site and a 378-bp fragment with a centrally
located EcoRI site (Figs. 5
and 6A). Using optimized
concentration of Mg2+ (31) we observed a distinct
difference in the cleavage rate of the long and short DNA fragments by
EcoRI endonuclease, ranging from 20 to 8% (Fig. 6,
A and B). This enhancement of the reaction rate,
proportional to the length of the DNA substrate, is usually explained
in terms of sliding or intersegment transfer (36). The sliding process
can be viewed as a one-dimensional diffusion of the nonspecifically
bound protein (totally electrostatic binding mode) along the DNA, which
is thought to occur by the displacement of bound (delocalized) positive
counterions from the DNA (36). Thus, any influence decreasing the
lifetime of the nonspecifically bound state (e.g. increase
of salt concentration (37)) or presenting a steric obstacle to linear
movement (e.g. nonsaturating amounts of histone-like protein
Hu (31)) will notably diminish the reaction rate dependence on the DNA
length. This was not the case for the inhibition of the
EcoRI cleavage reaction by THPs. On the contrary, we found
that the difference in the cleavage rate of the long and short DNA
fragments somewhat increased upon increasing THPs concentrations (Fig.
6), suggesting that the linear diffusion was not significantly affected
by THPs in the concentration range employed. Higher concentrations of
THP(A) led to very low levels of remaining endonuclease activity for
short DNA fragments to be quantified from a gel and compared with the
rate of cleavage of long DNA fragments.
THP(A) Affects EcoRI Binding to DNA--
It is known that
EcoRI endonuclease first binds to its recognition site and
then the enzyme-DNA complex binds Mg2+ (38). Thus, the
DNA-protein complex formation in the absence of Mg2+ can be
used to probe the effect of THP(A) on EcoRI binding to DNA.
The experiments were conducted with a minimal amount of
EcoRI endonuclease, needed for complete binding of the
d(CGCGAATTCGCG) duplex, and with a 5-fold excess of the
enzyme, and they exhibited similar results. As shown in Fig.
7, A and B, THP(A)
inhibited EcoRI-oligonucleotide complex formation, starting
at 1-1.5 mM concentrations, with an inhibition constant,
Ki (50% inhibition of binding), of 2.0-2.5
mM. Higher THP(A) concentrations, 3.5-4.0 mM,
provided complete (95-100%) inhibition. These data indicate that
THP(A) arrested the DNA cleavage by EcoRI as soon as the
first step of the reaction by preventing EcoRI binding to
its target DNA sequence.
Effect of Methylated Derivatives of THPs--
In order to
establish whether the zwitterionic nature of THPs is necessary for the
observed inhibition effect, we prepared carboxymethyl derivatives of
THPs (Fig. 1) and tested their effect on the reaction catalyzed by
EcoRI endonuclease. The ability of these compounds to
inhibit the endonuclease reaction was almost 100-fold lower, in
comparison to the respective THPs, and at low concentrations, the
methylated THP(B) derivative even enhanced the reaction rate as seen in
Fig. 3 (note the logarithmic scale). It is possible that the slight
inhibition effect was observed due to the minor amounts of unmethylated
THPs remaining in the preparation.
Effect of THP(A) on Several Type II Restriction
Endonucleases--
The inhibition of DNA cleavage (95-100%) by
THP(A) was also demonstrated for other type II restriction
endonucleases (Table I), in the range of
2-4 mM. Although the incubation buffers were different for
most of the enzymes used, we have not noticed any correlation of the
observed inhibition effect to the buffer composition. The finding that
THP(A) inhibits SgrAI restriction endonuclease was
unexpected, because both SgrAI and THP(A) originate from the same microorganism Streptomyces griseus (7). However, a few endonucleases appeared to be less sensitive to THP(A), e.g.
more then 90% of the remaining activity was detected at 50 mM THP(A) for SmaI endonuclease and about 53%
for PvuII. As SmaI is known to be a
thermosensitive enzyme, experiments with this endonuclease were
conducted at 30, 28, and 24 °C and showed similar results. To verify
whether a temperature lower than that of 37 °C, commonly used
throughout this study, can diminish the effect of THP(A), the assay for
EcoRI endonuclease was also conducted at 30 °C, with an
effect similar to that at 37 °C.
To assess whether the inhibition by THP(A) is dependent on the nature
of recognition sites, we further examined the effect of THP(A) on the
cleavage of DNase I, RNase A, and Taq Polymerase Reactions Are Not Affected by
THPs--
For the sake of comparison to endonucleases, we examined the
effect of THPs on the reaction catalyzed by DNase I, RNase A, and
Taq polymerase, enzymes interacting with nucleic acids in a
nonspecific manner. All three enzymes were extensively studied, and the
structures of their complexes with nucleic acids have been solved
(40-42). THP(A), THP(B), or the mixture of THP(A) and THP(B), as it
frequently appears in the microbial cell, up to 27 mM did
not affect the rate of the DNase I reaction. In addition, no changes in
the reactions catalyzed by RNase A or Taq polymerase were
observed in the presence of up to 60 and 500 mM THPs, respectively.
Spermine Restores DNA Cleavage and Binding by EcoRI, Arrested by
Moderate THP(A) Concentrations--
The finding that even the low
level of THP(A) in S. griseus at normal physiological
conditions is sufficient to inactivate SgrAI endonuclease
in vitro raised the question of how the restriction system
functions in bacteria that produce or accumulate this osmolyte. It is
well known that DNA in vivo is complexed with polyamines, which are essential components of many, if not all prokaryotic organisms (for a review, see Ref. 43). In E. coli,
concentrations of putrescine and spermidine range from 64 to 8 and from
6 to 18 mmol/liter of cytoplasmic water, respectively, depending on the
medium osmolarity (44). In mesophile Streptomyces bacteria (including S. griseus), in addition to putrescine and
spermidine, spermine is commonly detected (45). To assess whether
polyamines could be remedial for the restriction system inhibited by
THPs, we studied the effect of spermine on EcoRI binding to
and cleavage of ((dCGCGAATTCGCG))2 oligonucleotide,
arrested by THP(A). We found that spermine from a concentration of 0.1 mM starts to restore DNA cleavage inhibited by 5 mM THP(A), whereas at 3 mM, it already inhibits
the cleavage by itself (Fig.
9A). Fig. 9B shows
that spermine restores EcoRI binding to oligonucleotide,
starting from 0.1 mM, and leads to complete binding at 0.5 mM. Nevertheless, as seen in the Fig. 9B,
spermine only partially restores EcoRI binding to
oligonucleotide arrested by 10 mM THP(A).
Possible Mechanism(s) of THP Effect--
To elucidate the
molecular basis of the inhibition effect of THPs, the impact of these
compounds on every element of the endonuclease reaction has to be
considered. Our results reveal that THPs do not denature or modify the
restriction enzymes in the reaction mixture. This notion is also
supported by the findings that THPs can preserve activity of different
enzymes (46, 17). The possibility that THPs act by sequestering
magnesium ions in the reaction mixture can be ruled out. Furthermore,
THP(A) inhibits binding of EcoRI to DNA, for which
Mg2+ is not required. Our kinetics data indicate a
competitive type of inhibition, with Vmax
remaining constant and estimated Ki = 0.16 ± 0.04 mM. This type of inhibition suggests that the
inhibitor solely interferes with the enzyme-substrate complex
formation, without affecting the catalytic step of the reaction. This
notion was further supported by gel-shift analysis, which revealed
inhibition of EcoRI-oligonucleotide binding by THP(A) in
millimolar concentrations. A few mechanisms for THPs interference with
enzyme-DNA association are possible. The inhibitor can displace the
substrate from the active site of the enzyme, or recombine with the
substrate, or modulate energetic components of binding without direct
interaction with the enzyme or DNA. Direct binding of THP(A) to DNA is
unlikely, as was revealed by equilibrium dialysis and the absence of
effect on DNA melting.2 In contrast, a very weak
interaction with DNA was shown by a two-dimensional NMR study for
THP(B), along with lowering DNA melting temperature and elimination of
the dependence of DNA melting on base pair composition.2
Yet THP(B) destabilized the DNA duplex at much higher concentrations (1-4 M) than that needed to inhibit the endonuclease
reaction and was 10-fold less effective in inhibiting endonucleases
than THP(A), suggesting that effects other then DNA destabilization are
responsible for endonuclease inhibition by THPs.
To ensure fast location and binding to a specific site on DNA,
restriction endonucleases first bind nonspecifically anywhere to the
DNA and then scan the DNA in search of its recognition site in a
one-dimensional diffusion process (47, 48). The existence of sliding as
a mean of target location for DNA-binding proteins has been described
also in a number of other systems, such as transcription regulatory
proteins and RNA polymerase binding to its promoter site (36). In an
attempt to assess whether the steps of the enzyme association with DNA
can be inhibited independently or cooperatively by THP(A), we have
found that the linear diffusion of EcoRI, and consequently a
nonspecific binding as a prerequisite of linear diffusion, was
unaffected up to 1.5 mM THP(A) (Fig. 6, B and
C). Linear diffusion is dependent mostly on the
electrostatic interactions between the protein and the phosphate groups
of the DNA (47). Because of its electrostatic nature, nonspecific
binding is known to be strongly dependent on the salt concentration of the buffer. THPs most likely do not distort the electrostatic equilibrium to the same extent as do inorganic ions, which makes them
preferable to salts as intracellular osmolytes.
Insensitivity of enzymatic reactions catalyzed by proteins that bind
DNA nonspecifically, such as DNase I, RNase A, and Taq DNA
polymerase, to inhibition by THPs supports the above notion that these
compounds are "safe" to nonspecific protein-DNA interactions. It is
important to note that both DNase I (49) and RNase A (42) are
characterized by extensive interaction with the DNA phosphate backbone.
We further compared the effect of THPs within a number of type II
restriction endonuclease that differ in sequence of recognition site,
GC/AT ratio, cleavage pattern (blunt or overhang ends), etc. (Table I).
The observation that one out of three recognition patterns of
AvaI endonuclease on
As the structures of DNA-restriction endonuclease complexes have been
solved for only a few endonucleases (EcoRI,
EcoRV, BamHI, and PvuII) (47) and
additional indirect data on the DNA bending and interaction pattern in
specific complexes are available only for a limited number of
restriction enzymes, the extended screening for the susceptibility of
restriction enzymes to THPs inhibition might serve as a simple tool to
gain preliminary information on the structure of DNA-protein interface.
The overall standard binding free energy (
One of the contributions that is favorable for binding energetics
results from electrostatic coulombic interactions between positively
charged protein side chains and negatively charged DNA phosphates.
Coulombic forces are inversely proportional to the dielectric constant
of a medium and distances between two interacting charges. THP
molecules most likely increase the dielectric constant of the medium
like other zwitterionic compounds (25) and, if not repelled from
protein or DNA surface upon enzyme-DNA complex formation, may cause a
steric interference to the decrease of distances of charges
interaction. Both effects will eventually lead to a decrease in
favorable energetic contributions of protein-phosphate contacts. The
inability of the methylated THP derivatives to inhibit DNA cleavage by
EcoRI could be explained by the loss of zwitterionic structure characteristic for THPs and is in support of the above suggestion. The other major factor contributing favorably to binding energy is the release of bound water from nonpolar surfaces, as a
result of interactions between complementary regions of the nonpolar
surface "hydrophobic effect" (55). Thus, interference of THPs with
water release and restructuring of the water interface would also
decrease its favorable energetic contribution. This interference might
be crucial for the specific complexes, in which additional energy is
necessary for the DNA distortion by kinking, bending, and unwinding,
and interactions with specific phosphate groups play an additional role
in stabilizing a kinked DNA conformation in the protein-DNA complex.
This interference depends on whether THPs belong to the groups of
preferentially excluded or preferentially interacting solutes (56); in
other words, on the affinity of THPs to the protein surface in
comparison to that of water. For example, so called, solvophobic
compounds (one of the categories of the preferentially excluded) make
contact of the nonpolar regions of the protein with the solvent more
unfavorable thermodynamically than their contact with water, and thus
render the hydrophobic effect even stronger (56).
It was noted that most of the natural osmolytes belong to the class of
compounds that are protein stabilizers and are preferentially excluded
by a nonspecific mechanism, such as the increase in surface tension or
the solvophobic effect (56). Still, it is worth noting that the
delocalized positive charge of THPs is not characteristic of other
zwitterionic osmolytes, such as proline and betaine, nor is the ability
to interfere with protein-DNA interaction. In order to further refine
the mechanism of THPs interference with specific DNA-protein
interaction, data on the dielectric constant of THPs solutions and THPs
localization in the solution relatively to the macromolecules surface
(preferential exclusion or preferential interaction) has to be collected.
In Vivo Relevance of the Observed THP Effect--
Inhibition of
endonuclease-catalyzed DNA cleavage by DNA-binding ligands has been
described previously for a number of drugs, e.g. proflavin,
olivomycin, ethidium bromide, actinomycin D, and distamycin A (26). Yet
these inhibitors do not normally occur in microbial cells at
physiological conditions. Even in microorganisms producing antibiotics,
these metabolites are actively excreted from the cell to avoid suicide
(57). On the contrary, polyamines (putrescine, spermine, and
spermidine), prokaryotic histone-like proteins NS1 and NS2 (27), and
polyphosphate (28) are common bacterial cell constituents that inhibit
restriction endonuclease-catalyzed DNA cleavage. Now we can add THPs to
this list of substances. However, THPs exhibit a number of distinct
features compared with those of polyamines: (i) DNA cleavage by
restriction endonucleases is not activated by low concentrations of
THPs as it is by spermine and spermidine; (ii) although polyamines bind
to double-stranded DNA and stabilize it, binding of THPs to DNA was not
detected, and THP(B) significantly destabilized DNA; (iii) THPs do not
cause any apparent condensation or precipitation of DNA as do
polyamines at concentrations of 0.2-0.3 mM (27). The
intracellular concentration of THP(A), as shown for a number of
Streptomyces strains, ranges from 15-20 mM at
normal growth conditions to 50-150 mM as a response to
osmotic stress or to 38-44 mM at elevated temperatures
(7). The finding that THP(A) inhibits SgrAI endonuclease in
the range of 2-4 mM, similarly to other tested
endonucleases, suggests that the restriction enzymes of the
Streptomyces bacteria are not resistant to the inhibition by
THP(A). Such a coexistence of restriction enzyme and its inhibitor in
concentrations sufficient to completely suppress DNA cleavage in
vitro raised the question of how the functions of restriction
system are retained. We have shown that spermine (and most likely other
polyamines) can compensate the inhibition of EcoRI binding
and cleavage of DNA, by moderate concentrations of THP(A). Yet THP(A)
concentrations higher than 10 mM were compensated only
partially by spermine, suggesting that THPs still can lead to
alleviation of restriction in vivo. Indeed, heat sensitivity of the restriction system in vivo has been reported for a
number of Streptomyces strains (58, 59) and for
Corynebacterium glutamicum (60). It is tempting to propose
that the alleviation of restriction in these cases may be the result of
induction of the intracellular synthesis of THPs under heat stress. It
was suggested by Schafer et al. (60) that under stress,
restriction would be alleviated, and the cell could acquire foreign
genetic information more easily, possibly enhancing the capability to
deal with the particular environmental requirement. In addition, high
endonuclease activity is lethal for bacteria, if it is not balanced by
the activity of corresponding methylase and DNA ligase (21); therefore,
the restriction alleviation might also be a part of bacterial survival strategy under stress. Finally, the potential ability of THPs to
alleviate restriction and to be efficiently taken up by bacteria (7, 9)
may be of use in genetic manipulation with microorganisms. However, the
correlation of the effect of THPs on restriction endonuclease activity
in vitro, and the modulation of the restriction system
in vivo needs to be further investigated.
DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide
(d(CGCGAATTCGCG))2 at about 4.0 mM.
THP(B) was 10-fold less effective than THP(A), whereas for betaine and
proline, a notable inhibition was observed only at 100 mM.
Similar effects of THP(A) were observed for all tested restriction
endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of
THP(A) on the activity of DNase I, RNase A, and Taq DNA
polymerase was noticed. Gel-shift assays showed that THP(A) inhibited
the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI
along the DNA was not affected. Methylation of the carboxy group
significantly decreased the activity of THPs, suggesting that their
zwitterionic character is essential for the inhibition effect. Possible
mechanisms of inhibition, the role of THPs in the modulation of the
protein-DNA interaction, and the in vivo relevance of the
observed phenomena are discussed.
INTRODUCTION
Top
Abstract
Introduction
References
charge in the NCN group (Fig. 1) and
form the half-chair conformation (8).
MATERIALS AND METHODS
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Fig. 1.
The molecular formulae of THP(A), THP(B), and
their methyl derivatives.
-32P]ATP (6000 Ci/mmol, New England
Biolabs) by T4 polynucleotide kinase (30), purified by denaturing
polyacrylamide gel, and desalted on Sephadex G-25. The
32P-labeled oligonucleotide was heated for 5 min at
90 °C in a buffer containing 20 mM Tris-HCl (pH 7.4),
100 mM NaCl, 10 mM MgCl2, 0.1 mM dithiothreitol and then slowly cooled to 4 °C,
desalted on Sephadex G-25, purified by step elution on DEAE-Sephadex
(10 mM bis-Tris propane, pH 7.4), with increasing
concentrations of KCl (up to 0.6 M KCl) and desalted by
dialysis against water. About 95% of the resulting oligonucleotide was
cleavable by EcoRI endonuclease.
-mercaptoethanol; 0.1 mg/ml bovine serum albumin; 10 nM [
-32P]dATP; 1.0 µM dATP;
dCTP, dGTP, and dTTP (200 µM each); 0.6 mM activated calf thymus DNA (Sigma); 40 units/ml of Taq DNA
polymerase; and different concentrations of THPs. After 15 min
incubation (corresponding to the region of linear reaction kinetics, as
determined in a separate experiment). The reaction was stopped by
addition of 10 µl of 50 mM EDTA and applied to strips of
chromatographic paper (Whatman No. 3). Strips were washed three times
with ice-cold trichloroacetic acid and dried, and the radioactivity was
measured by a scintillation counter.
RESULTS
linear DNA.
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Fig. 2.
Cleavage of pBR322 DNA by
EcoRI endonuclease in the presence of THP(A).
A, pBR322 DNA (25 µg/ml) was incubated with 125 units/ml
EcoRI in 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCl in the
absence and presence of THP(A), as indicated. Reaction products were
analyzed on a 1% agarose gel. The positions of supercoiled
(sc), linear (li), and nicked (ni) DNA
are indicated. B, Dixon plot of pBR322 DNA cleavage by
EcoRI in the presence of THP(A). The data points for each
DNA concentration are the average of at least three independent kinetic
experiments. , 2 nM pBR322 DNA;
, 4 nM;
, 6 nM. C, Cornish-Bowden plot of the data
presented in B.
, 2 nM pBR322 DNA;
, 4 nM;
, 6 nM.
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Fig. 3.
Effect of osmolytes and methyl derivatives of
THPs on the rate of pBR322 DNA cleavage by EcoRI.
Every data point reflects the initial rate of cleavage (the region of
linear reaction kinetics has been determined in a separate experiment)
of pBR322 DNA (37 µg/ml) by EcoRI restriction endonuclease
(37 units/ml) in the presence of the corresponding solute, normalized
by the rate of cleavage in the absence of additives. , THP(A);
,
THP(B);
, methyl-THP(A);
, methyl-THP(B);
,
L-proline;
, glycine betaine. Data points are
the average of at least two independent experiments.
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Fig. 4.
Cleavage of
[32P](d(CGCGAATTCGCG))2 by
EcoRI endonuclease in the presence of increasing
concentrations of THP(A). A, autoradiograph of products
of [32P](d(CGCGAATTCGCG))2 (1.0 nM) cleavage by EcoRI endonuclease (0.15 units/µl) on denaturing polyacrylamide gel; B, inhibition
curve of the cleavage reaction, calculated as a ratio of cleaved to
total DNA, obtained from the densitometry of polyacrylamide gel
autoradiograph presented in A. The reaction was carried out
as described under "Materials and Methods."
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Fig. 5.
Graphic map of the pBR322 plasmid used for
preparation of the substrate for probing the facilitated diffusion of
EcoRI endonuclease. The pBR322 plasmid DNA was
cleaved either by PvuII endonuclease, yielding the linear
fragment of 4361 bp, or simultaneously by SspI and
EcoRV endonucleases, producing a fragment of 378 bp with a
unique EcoRI site in the central position.
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Fig. 6.
Effect of THPs on the
EcoRI-catalyzed cleavage of long and short DNA
fragments. 97 nM pBR322 plasmid DNA predigested with
SspI/EcoRV (yielding the short DNA fragment with
an EcoRI site) or PvuII (producing a long DNA
fragment with an EcoRI site) was incubated in 15 µl of
reaction mixture with 50 units/ml EcoRI and THP(A) or THP(B)
alone added as indicated at 37 °C. After 25 min of incubation
(corresponding to the region of linear kinetics) the reaction was
terminated by addition of 10 mM EDTA, and the reaction
products were separated by electrophoresis. A, agarose gel
electrophoresis of EcoRI digest upon addition of THP(A);
B, densitometry analysis of the gel shown in A
reflects the inhibition of cleavage of long ( ) and short (
) DNA
fragments by THP(A); C, inhibition of cleavage of long (
)
and short (
) DNA fragments by THP(B). Data points are mean values
from at least two independent experiments.
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Fig. 7.
Inhibition of
EcoRI-(d(CGCGAATTCGCG))2 complex formation
by THP(A). A, electrophoretic mobility of
EcoRI-dodecanucleotide complexes in the presence of
increasing concentrations of THP(A) in nondenaturing polyacrylamide
gel. (d(CGCGAATTCGCG))2 (0.1 nM) was incubated
with 5 units of EcoRI in 20 µl of binding buffer;
B, inhibition curve, calculated as a ratio of bound to total
DNA, obtained from the densitometry of the polyacrylamide gel
autoradiograph presented in A. The enzyme-DNA complex
formation was followed by electrophoresis as described under
"Materials and Methods."
Restriction endonucleases, used throughout the study, and their
recognition sites
phage DNA by AvaI restriction endonuclease. AvaI has eight recognition sites on
phage DNA, CCCGGG
(three sites), CCCGAG (two sites), CTCGGG (two sites), and one site of the CTCGAG sequence. It appeared that not all of these sequences were
equally sensitive to the inhibition by THP(A). The appearance of the
band of 6500 bp (Fig. 8) may indicate the
preferential inhibition of the cleavage of the CTCGAG recognition site
of AvaI. Alternatively, this observation may reflect that
AvaI cleaves CTCGAG with the lowest rate as compared with
the other seven sites, although such a hierarchy of sites was not
observed previously for this enzyme (39). It is worth noting that this
sequence is characterized by the lowest GC content among the eight
others, whereas the highest GC content has the sequence identical to
the recognition site of SmaI (Table I), although
SmaI produces DNA fragments with blunt ends, comparing to
overhang ends for the AvaI.
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Fig. 8.
Effect of THP(A) on the cleavage of DNA by AvaI restriction endonuclease.
DNA (1 µg) was cleaved by 6 units of AvaI restriction
endonuclease (minimal amount of enzyme that led to complete cleavage in
1 h), in 10 µl of the reaction mixture. The arrow indicates the
position of the band which appear as a result of preferential
inhibition of
DNA cleavage by AvaI at its CTCGAG
recognition site.
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Fig. 9.
Spermine compensation of the THP(A) effect on
DNA cleavage and binding by EcoRI. A,
restoration of pBR322 DNA (1 µg) cleavage (2.5 units of
EcoRI endonuclease in 20 µl of the reaction mixture),
arrested by 5 mM THP(A), by spermine tetrahydrochloride.
The reaction was initiated by addition of the enzyme. The reaction
products were analyzed on a 1% agarose gel. The positions of
supercoiled (sc), linear (li), and nicked
(ni) DNA are indicated. B, restoration of
(d(CGCGAATTCGCG))2-oligonucleotide binding to
EcoRI endonuclease by spermine. Positions of the bound and
free oligonucleotide are indicated. The gel retardation experiment was
conducted essentially as in Fig. 7B. Spermine was added to
the reaction mixture prior to the addition of the EcoRI
endonuclease.
DISCUSSION
DNA, was preferentially inhibited by THP(A) suggested that inhibition may be
sequence-dependent. Another striking observation was that
PvuII and SmaI endonucleases were much less
sensitive to THPs inhibition effect than all other restriction enzymes
studied. What are the features of SmaI and PvuII
interaction with DNA that make them respond differently than other
restriction endonucleases tested for inhibition by THPs? Withers and
Dunbar (50) noticed that PvuII displays certain similarities
with SmaI, such as (i) interaction with each of the base
pairs within the recognition site, (ii) a potential protein contact to
the phosphate 3' to the scissile bond, and (iii) no significant bending
of the DNA. In addition, SmaI interactions with DNA
phosphates are all adjacent and clustered within the recognition site
(50), whereas EcoRV endonuclease exhibit phosphate interactions both within and flanking the recognition sequence (51). In
EcoRI, DNA phosphate interactions are delocalized, so the
primary clamp occurs at the immediate 5'-end of the recognition sequence, and supplementary clamps occur adjacent to the primary and at
the center of the recognition sequence (52). The most remarkable common
feature of SmaI and PvuII is that the
PvuII does not distort DNA while bound to its recognition
sequence (53), and SmaI bends DNA insignificantly (50). This
distinguishes them from the other well studied endonucleases,
e.g. upon binding to canonical DNA sites, both
EcoRI and EcoRV endonucleases appear to drive the
DNA into a rather unfavorable conformation (51). Sequence specific
phosphate contacts in the EcoRI-substrate complex anchor and
orient protein recognition elements within the major groove of the DNA,
and they also act as clamps to stabilize the kinked DNA conformation in
the complex. Moreover, the interference with any one of these three
phosphates (six per duplex) causes a large effect on binding energy and
must reflect the cooperative loss of other contacts (52).
G°bind) is
the net of favorable and unfavorable energetic contributions. In the case of EcoRI endonuclease, these contributions were
recently summarized for both the "specific" recognition complex at
the GAATTC site and the nonspecific complex (54). Consequently, the net
free energy of specific binding includes favorable contributions from
protein contacts with DNA bases, phosphate contacts, and release of
bound water from nonpolar surfaces and unfavorable contributions from
restriction of rotational and translational freedom of the protein and
DNA, DNA distortion, protein conformation, etc. (54).
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ACKNOWLEDGEMENTS |
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We greatly appreciate the helpful assistance of Dr. Edna Ben-Asher in the first stages of this study. We also thank Alexander Litovchick for assistance in preparation of THP(A) and Dr. Jack Cohen for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in parts by grants from the Israel-United States Binational Science Foundation, the European Commission-Israel Ministry of Science and the Arts, and the Israel Academy of Science (to A. L.).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 in part by a Hirsch and Faine Raskin Foundation, Inc., scholarship.
§ To whom correspondence should be addressed. Tel.: 972-8-9343-413; Fax: 972-8-9344-142; E-mail: colapidot{at}wiccmail.weizmann.ac.il.
2 R. S. Iakobashvili, G. Malin, and A. Lapidot, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: THP(A), 2-methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine; THP(B), 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine; THP, tetrahydropyrimidine derivative; bp, base pair(s).
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REFERENCES |
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