(Received for publication, July 25, 1995; and in revised form, September 8, 1995)
From the
A series of modified nucleotides was used to map the hydrogen-bonding and hydrophobic sites of the TerB DNA required for Tus interaction. Each of four consensus guanine residues in the TerB-binding site was replaced by 7-deazaguanine, 2-aminopurine, or inosine nucleobase analogues, and each thymine by a uracil analogue. The observable equilibrium dissociation constant for the Tus protein-TerB DNA complex was measured at pH 7.5, 25 °C, and 150 mM potassium glutamate using a competition binding method. Substitutions made at position 10 with a 7-deazaguanine, 2-aminopurine, or inosine analogue had a large effect on the stability of the complex, approximately +3 kcal/mol in each case. Substitutions made at positions 13 and 17 had a varied response. For uracil substitutions, potential hydrophobic sites were identified at six positions in the TerB DNA. The energetic penalty for the removal of a single methyl group ranged between +1 and +2 kcal/mol. Rate dissociation measurements agree with these results. Overall, major and minor groove determinants are required for binding. An unusual result was that the conserved nucleotide at position 6 did not significantly affect in vitro binding of the complex.
In Escherichia coli, termination of replication is
mediated by the specific interaction of a protein called Tus with a
series of DNA sequences known as Ter. The Tus protein binds as
a single subunit to its DNA-binding site (1, 2) and
does not appear to have homology to any of the known DNA-binding
motifs(3) , although some similarities have been noted with
some DNA- or RNA-binding proteins(4) . A comparison of all Ter sites found on the chromosome and on plasmids revealed an
11-nucleotide sequence that is absolutely conserved for all
sites(5) . Despite the relatively small number of nucleotides
that constitute the Ter-binding site, the observable binding
constant for a 37-base pair fragment containing a TerB-binding
site is 3.3 10
M
in
150 mM potassium glutamate, pH 7.6. The measured dissociation
rate for this site is 2.1
10
s
, a half-life of 550 min(6) .
The in vitro activity of the Tus-Ter complex impedes the progress of reconstituted replication forks on both the chromosome and the plasmids of E. coli(7, 8) . This activity is orientation-dependent and is consistent with the asymmetric binding of this protein(2, 6) . The action of this complex and its ability to impede replication forks are associated with anti-helicase properties reported under some conditions(7, 10) . The mechanism of action of this protein has not been elucidated, although two possibilities have been proposed(6) . Either the protein-DNA complex produces a thermodynamically stable barrier to oncoming replication forks, or protein-protein interactions are required for stalling replication forks. Recently, evidence supporting the latter mechanism was reported(11, 12) .
Modified nucleotides are an increasingly useful means of investigating interactions between proteins and DNA. Isosteric analogues inserted into the DNA report on the relative importance of specific groups required by the complex for recognition and binding. In this work, we have used a series of modified nucleotides to analyze the Tus-TerB interaction. Two G analogues (2-aminopurine and 7-deazaguanine) were used to probe major groove sites, and a third analogue (inosine) was used to probe minor groove sites of G. A fourth analogue (uracil) allowed us to test the relative importance of methyl groups of T in the TerB binding sequence. From these results, we have mapped some of the chemical groups in the major and minor grooves of the TerB-binding site that are determinants for the interaction of Tus protein binding. Because we were able to measure the extent of the perturbation to the complex, our results show that the DNA major and minor grooves situated between nucleotides 8 and 19 are crucial for the interaction, although there are minor contributions from other nucleotides outside this region. Of particular interest is the need for six methyl groups that help stabilize the complex, presumably through van der Waal interactions. These measurements also demonstrate that analogue substitutions at position 6 do not appreciably affect the complex, and therefore, it is not essential for Tus binding in vitro.
In the competition binding
assay, a constant amount of labeled wild-type TerB (1
10
M) was incubated with the Tus protein
(1.8
10
M) and competing amounts
of analogue DNA. All protein and DNA dilutions were done in binding
buffer containing 50 mM Tris-Cl, pH 7.6 (at 25 °C), 150
mM potassium glutamate, 0.1 mM dithiothreitol, 0.1
mM EDTA, and 100 µg/ml bovine serum albumin. The total
volume of the samples for this assay was 800 µl. The samples were
incubated for at least 3 h. 500 µl of each sample were filtered
through nitrocellulose filters presoaked in wash buffer (50 mM Tris-Cl, 150 mM potassium glutamate, and 0.1 mM EDTA) and washed with 650 µl of wash buffer. Background counts
for each assay were determined by incubating 600 µl of wild-type
DNA, 100 µl of competitor DNA, and 100 µl of binding buffer.
The total quantity of Tus protein available for binding was determined
by incubating saturating quantities of Tus protein with wild-type TerB DNA only. Each assay was repeated at least three times.
Errors in the filter assay due to the loss of material were not
corrected by the recently developed method of Wong and Lohman (18) .
The results were fit to a binding curve using nonlinear regression analysis with the graphing program SIGMAPLOT (Jandel Scientific) using :
On-line formulae not verified for accuracy
where R, K
, K
, C
, and O
are the total protein, equilibrium dissociation constant for
wild-type operator, equilibrium dissociation constant for competitor,
total competitor DNA, and total labeled operator, respectively. The
binding constant derived for the wild-type Tus-TerB complex
using the competition method was identical to the value obtained by the
standard filter binding assay previously determined(6) .
On-line formulae not verified for accuracy
where cpm is the number of counts bound to the filter at time t, cpm is the counts at time 0, k
is the dissociation rate constant, and bkg is the background
counts.
Figure 1: Sequence of the duplex DNA used for determination of the equilibrium dissociation constant. The consensus sequence of the TerB site is in boldface and is underlined. The numbering system used for nucleotide identification is indicated above the sequence.
Figure 2: A, chemical structures of the isosteric nucleotide analogues used in this work. Arrows indicate sites of modification. Two analogues (7-deazaG and 2-aminopurine) introduce modifications in the major groove. The former analogue has the nitrogen at position 7 replaced by a carbon, and the 2-aminopurine nucleotide has the carbonyl group of guanine removed with deprotonation of N-1. A third analogue (inosine) has the amino group at position 2 removed and is considered a probe for minor groove interactions. B, the analogue 2-aminopurine is shown paired with either cytosine or uracil. Pairing with cytosine gives rise to the protonated species, which are in the equilibrium form depicted above.
Substitution with the analogue 7-deazaG
affected the free energy of binding at positions 10, 13, and 17 and not
at position 6 within experimental error ( Fig. 3and Table 1). The free energy change at position 10 was the largest
at 3 kcal/mol, while modifications at positions 13 and 17 were
1 kcal/mol. For substitutions containing the 2-AP analogue, the
binding constants for TerB modified at position 10 or 13 were
significantly different from the wild type, with
G
= 3.1 and 2.9 kcal/mol,
respectively ( Fig. 3and Table 1). The position 17
substitution had a measured free energy change of
0.7 kcal/mol,
while the 2-AP nucleotide at position 6 resulted in only a small effect
on the binding constant (Table 1).
Figure 3:
Determination of the observable binding
constant for the Tus protein with analogues that test for potential
hydrogen-bonding sites in the major or minor groove. The data represent
7-deazaguanine, 2-AP paired with cytosine or uracil, and inosine that
were incorporated into the TerB-binding site at the indicated
sites. All measurements were made in 50 mM Tris-HCl, pH 7.6
(at 25 °C), 150 mM potassium glutamate, 0.1 mM dithiothreitol, and 100 µg/ml bovine serum albumin using a
nitrocellulose competition binding method. The results were plotted,
and the line through the data is the best theoretical fit according to . Blk, cpm in the absence of competitor DNA.
, wild type;
, 2-AP
C (position 10);
,
2-AP
C (position 13);
, 2-AP
U (position 10);
,
2-AP
U (position 13);
, 7-deazaG (position 10);
,
7-deazaG (position 13);
, 7-deazaG (position 17);
, inosine
(position 10);
, inosine (position
17).
Figure 4:
Determination of the observable binding
constant for the Tus protein using a nitrocellulose competition binding
method. Potential hydrophobic sites were identified by the insertion of
uracil instead of thymine in the TerB-binding site. Labeled
wild-type TerB DNA at a concentration of 1
10
M was added to the indicated
concentration of competitor and allowed to equilibrate with the Tus
protein (1.8
10
M) in the
following buffer: 50 mM Tris-HCl, pH 7.6 (at 25 °C), 150
mM potassium glutamate, 0.1 mM dithiothreitol, and
100 µg/ml bovine serum albumin. The results were plotted, and the
line through the data is the best theoretical fit according to . Blk, cpm in the absence of competitor DNA.
, wild type;
, uracil (position 9);
, uracil
(position 12);
, uracil (position 14);
, uracil (position
8);
, uracil (position 16);
, uracil (position 19);
, uracil (positions 12/16).
Figure 5:
Comparison of dissociation rates for the
Tus protein from wild-type TerB and three different
substituted TerB sites using isosteric analogues for the
guanine residues. Typically, the Tus protein (1.5
10
M) was equilibrated with DNA at a
concentration of 2
10
M. Excess
unlabeled DNA was added, and aliquots were removed at the indicated
times. Measurements were made in 50 mM Tris-HCl, pH 8.0 (at 25
°C), 150 mM potassium glutamate, 0.1 mM dithiothreitol, and 100 µg/ml bovine serum albumin. The
results were plotted, and the line through the data is the best
theoretical fit according to .
, wild type;
,
7-deazaG (position 13);
, 7-deazaG (position 17);
,
2-AP
C (position 17);
, 2-AP
U (position 10);
,
inosine (position 17).
Figure 6:
Comparison of dissociation rates for the
Tus protein from wild-type and uracil-substituted TerB sites.
Typically, the Tus protein (1.5 10
M) was equilibrated with DNA at a concentration of 2
10
M. Excess unlabeled duplex DNA
was added, and aliquots were removed at the indicated times.
Measurements were made in 50 mM Tris-HCl, pH 8.0 (at 25
°C), 150 mM potassium glutamate, 0.1 mM dithiothreitol, and 100 µg/ml bovine serum albumin. The
results were plotted, and the line through the data is the best
theoretical fit according to .
, wild type;
,
uracil (position 9);
, uracil (position 12);
, uracil
(position 14);
, uracil (position 8);
, uracil (position
16);
, uracil (position 19).
The Tus protein is responsible for the termination of DNA replication in E. coli. The Tus protein interacts as a single subunit with Ter DNA and does not appear to have any symmetry, and the complex appears to function in a single orientation. Although we have used chemical protection methods to identify sites of interaction(6) , this approach does not provide enough information about the relative importance of functional groups and is limited by the reactivity of the reagent to specific chemical groups of the DNA. If the mechanism of inhibition of replication by the Tus-Ter interaction is to be elucidated, then the relation between the complex's stability and the termination event is essential. We therefore used a series of nucleotide analogues that gives us information about the relative contributions made by specific functional groups and also allows us to test DNA sites that are not amenable to study by other methods. Furthermore, the effects of these modified nucleotides on the protein-DNA complex are useful for comparing the Tus-TerB interaction with other protein-DNA complexes that have been tested with similar analogues.
Chemical
modification techniques have defined general features of the
DNA-binding site(6) . Dimethyl sulfate protection was observed
at the four guanine residues between and including positions 6 and 17.
The strongest protection sites were located at positions 10 and 13 in
the DNA. Based on this information, we reasoned that interactions
between the protein and the DNA are strongly indicated at positions 10
and 13 and that a protein segment might interact with N-7 of guanine
residues at positions 6 and 17 as well(6) . Our current
measurements have confirmed the necessity of specific chemical groups
at positions 10 and 13. The large energetic penalty of 3 kcal/mol,
brought about by the insertion of 2-AP at these two positions, clearly
indicates that the carbonyl oxygen is required for complex formation.
The substitution of 7-deazaG further underscores the role of the G
residue at position 10. Two other sites (positions 13 and 17)
contributed as well, but to a lesser extent, and are in agreement with
the protection studies. When we tested for minor groove participation
using inosine, once again, the position 10 substitution destabilized
the complex (
G
= 3.0 kcal/mol) as
well as the position 17 substitution (
G
= 1.7 kcal/mol).
The most surprising effect occurred at
position 6. We anticipated that replacing the guanine residue at
position 6 with 7-deazaguanine or 2-aminopurine nucleotides, a position
that is conserved in all known Ter site sequences and is
partially protected in chemical footprinting studies, would destabilize
the complex. However, the complex was little affected by the presence
of any of the modified nucleotides (G
= 0.3 kcal/mol). The combined data of the analogues
indicate that a unique base pair is not needed at this site.
Apparently, chemical modification techniques, while providing
information about the location of the protein on the DNA, are not
reliable enough for identifying site-specific interactions in the
protein-DNA complex. The question remains how a nucleotide position can
be conserved and yet not significantly contribute to the protein-DNA
complex's stability. One possibility is that the mechanism of
termination requires weak interactions at this position that assist in
orienting the protein properly to impede translocating proteins.
Another possibility is that an additional factor is necessary to
modulate the activity of the Tus protein that may interact with this
conserved nucleotide and the Tus protein when positioned on the DNA (31) .
To confirm the requirement of both major and minor
groove interactions, we inserted a purine analogue at position 10. The
purine analogue has both the O-6 carbonyl and the amino group at
position 2 removed and is a structural combination of both the 2-AP and
inosine analogues. We reasoned that if a significant effect on the
binding resulted from structural perturbation to the DNA, then the
insertion of the purine analogue should alter the free energy very
little in comparison with either 2-AP or inosine analogues at this
position. On the other hand, if the free energy change is a combination
of the individual binding energies, then each group would contribute
independently to the formation of the bimolecular complex. Our
measurements showed that the insertion of purine at this site resulted
in a free energy change of >6.0 kcal/mol, supporting the independent
contributions of major and minor groove sites to the interaction. ()At this stage of analysis, we are unable to determine the
origin of the perturbations to the complex. If they reflect steric
clashes between the protein and DNA in the complex, then they appear to
exceed estimates that are currently assumed for such disruptions
(1.5-2.0 kcal/mol).
The use of uracil has served to further delineate methyl groups in the major groove essential for stabilizing the complex through ``hydrophobic'' interactions. Of all the sites tested, six proved to be critical for full binding activity, ranging in value from 1.0 to 1.9 kcal/mol (Table 2). These sites are dispersed through this entire region, with three sites in the top strand and three sites in the bottom strand. Three of these sites (positions 12, 14, and 16) are located in the major groove region, where critical hydrogen-bonding sites are shown in this work. The need for six methyl groups to stabilize the complex through van der Waal contacts is unusual and may explain why the Tus-TerB complex is able to achieve tight binding within a relatively small segment of DNA.
By comparing the G
value for
2-AP paired with cytosine to ones paired with uracil, we are able to
draw some conclusions about the effect of 2-AP on complex stability (Fig. 2B). The use of uracil instead of cytosine in the
complementary strand potentially affects the stability of the complex
in either of two ways. First, the introduction of uracil allows a
better match between the base pair donor and acceptor hydrogen bonds (Fig. 2B). A recent study using 2-AP paired either with
cytosine or uracil has demonstrated that, in general, the latter base
pair leads to a thermodynamically more stable duplex DNA(19) .
The increased DNA stability may result in a more stable protein-DNA
complex, as has already been demonstrated for the lac repressor-operator interaction(20) . The position 10
substitution of 2-AP
U for 2-AP
C, which improved the binding
of the Tus-TerB complex by approximately -0.7 kcal/mol,
would appear to represent this effect. Although we are unable to
precisely define the thermodynamic origin of the negative free energy,
namely the reconstitution of wild-type requirements for binding or the
introduction of new compensating effects, these results nonetheless
establish that the complex is partially destabilized by DNA structural
perturbations at this site.
On the other hand, the introduction of a carbonyl instead of an amino group may serve to further destabilize the protein-DNA interaction by removing a hydrogen bond donor of cytosine and replacing it with a hydrogen bond acceptor of uracil, directly perturbing the protein-DNA interface. An example of this is the substitution of uracil for cytosine at position 13, which resulted in an additional loss of free energy of 1.5 kcal/mol. This indicates that the amino group of cytosine is important for the formation of the complex, and its replacement in the complex offsets any possible gains made by stabilizing the DNA structure. The combined results of 2-AP paired with either cytosine or uracil imply a direct interaction along the protein-DNA interface in the major groove between positions 10 and 13, which is disrupted by the alignment of sterically incompatible groups.
The results from the substitution experiments are consistent with mutagenesis experiments performed with other Ter sites modified by single natural base pair mutations at positions 10, 12, 13, and 14, which do not bind the Tus protein and consequently are incapable of functioning as replication arrest sites. However, Tus is able to bind and arrest replication at Ter sites containing single substitutions at positions 6, 8, 11, 16, and 18(2) . These data, on a whole, reinforce our earlier prediction that the central major groove region defined by bases 10-13 is directly involved in the interactions with the Tus protein.
In those cases where measurement of the dissociation rate was possible, the values for the dissociation rate paralleled those obtained from equilibrium measurements ( Fig. 5and Fig. 6and Table 1and Table 2). From these results and for this size fragment, analogues that affect binding do so by altering the dissociation rate, and characterization of this constant when possible is sufficient for characterizing the complex.
There are now a number of systems, both
enzymatic and nonenzymatic, that have been analyzed using modified
nucleotides(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) .
In those cases that allow for direct comparison of equilibrium
dissociation constants, the free energy changes for the association
process for the two purine isosteric analogues used in this study were
in the range of 1.5-2.0 kcal/mol for the trp repressor and the EcoRI restriction
endonuclease(9, 25, 30) . Recently, we
measured the effect of 2-aminopurine and 7-deazaguanine on the lac repressor-operator interaction(20, 29) . In this
interaction, the analogue 7-deazaG substitutions perturbed the complex
between 1.5 and 2.5 kcal/mol, while the 2-AP substitutions ranged
between
1.0 and 3.0 kcal/mol. The free energy values obtained for
the Tus-TerB complex are similar to those of the lac repressor-operator, despite the different cations used to measure
the stability of the two protein-DNA complexes.