Volume 2 number 10 October 1975
N UCleiC Acids Research
Physical studies of the interaction between the Escherichia coli DNA binding
protein and nucleic acids
Ian J. Molineux+, Andrew Pauli, and Malcolm L. Gefter
Department of Biology, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA
Received
18 August 1975
ABSTRACT
The interaction of nucleic acid with the Escherichia coli
DNA-binding protein has been studied by fluorescence emission
spectroscopy and sedimentation velocity analysis. The protein
binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 x 10"^. It binds to the homopolymers poly(dA)
and poly(dT) slightly more tightly, but has a larger apparent
equilibrium dissociation constant to poly(dC). The protein also
binds tightly to ribohomopolymers and to tRNA, but not to duplex
DNA. By the use of defined-length oligonucleotides, it has been
shown that the protein binds to DNA in a highly cooperative
manner. The extent of cooperativity is seen as the difference
in binding between an isolated monomeric protein molecule bound
to DNA and two or more molecules binding to contiguous sites.
INTRODUCTION
The purification of the DNA-binding protein isolated from
uninfected E_. coli was greatly facilitated by the observation
that it eluted from denatured-DNA-cellulose only at a concentration of NaCl of two molar
, but it had little or no affinity
for native-DNA-cellulose.
The binding of this protein to single-strand DNA (SS-DNA)*
was shown by electron microscopy to be highly cooperative; i. e.,
when using excess DNA over binding protein, DNA was either toally
complexed with, or devoid of, the protein.
In addition, it was
shown that high concentrations of protein catalyzed the partial
denaturation of bacteriophage X
native DNA in a manner compar-
able to the partial denaturation observed in alkaline solution .
•Abbreviations used: SS-DNA = single-strand DNA; K^iss
equilibrium dissociation constant; k o f f = rate constant for
dissociation.
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It was suggested that the protein expanded denaturation loops,
as opposed to initiating new ones and that protein, although
lacking specificity for nucleotide sequences, preferentially
bound to the A-T rich region, i. e., those most easily denatured.
We have previously shown that in addition to the binding
protein binding to SS-DNA, the protein also interacts with E_.
coli DNA polymerase II and exonuclease I and with the T7-induced
DNA polymerase
'
to form specific protein complexes,
Further-
more, these DNA polymerases (but not exonuclease I) form
ternary complexes consisting of DNA, binding protein, and
enzyme.
Neither the binary nor the ternary complexes are as
stable to high ionic strength as a DNA: DNA-binding protein complex is; their formation is inhibited at 0.1 M KC1.
To gain
insight as to the biological significance of these complexes,
we have first examined some of the physical parameters associated
with the interaction of nucleic acid and binding protein.
We
took advantage of the fact that binding protein in solution
exhibits a characteristic fluorescence emission spectrum and
that this emission is quenched when the protein is bound to
nucleic acid.
The sensitivity of the technique allowed the
calculation of the apparent equilibrium dissociation constant
(K,.
) for the complex. .In addition, we have calculated the
stoichiometry of binding of the protein to SS-DNA, and, by the
use of defined-length oligonucleotides corresponding to one and
two protein monomer binding sites, we have measured the degree
and type of cooperativity of binding exhibited by the protein.
Furthermore, by velocity sedimentation of binding protein-DNA
complexes, we have confirmed the values for stoichiometry obtained by fluorescence spectroscopy and, in addition, measured a
minimal rate constant (k -,) and half life of the dissociation
reaction.
MATERIALS AND METHODS
A.
Nucleic acids
SS-DNA was isolated from bacteriophage fd by phenol-SDS
extraction and further purified by sedimentation through a linear
sucrose gradient (5-20% w/v) in 0.3 M NaOH-0.7 M NaCl-0.005 M
EDTA.
The UV-absorbing material sedimenting at 27S (fd unit-
length DNA) was pooled and dialyzed against 0.01 M Tris HC1
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pH 7.6, 0.001 M EDTA.
Double-strand DNA was isolated from
Salmonella phage P22 by gentle phenol extraction followed by
passage through nitrocellulose in 1 x SSC.
tRNAg
n
was obtained
from Boehringer Biochemical Corporation and was used without
further purification.
Polydeoxythymidylic acid (poly(dT)) and
polyadenylic acid (poly(dA)) were from Miles Laboratories.
The
oligodeoxythvmidylic acids of nucleotide chain length 16 and 8
((pT) 16 and (pT) g , respectively) were from PL Laboratories.
Polydeoxycytidylic acid (poly(dC)), polyribocytidylic acid
(poly(rC)), polyriboadenylic acid (poly(rA)), and polyribouridylic acid (poly(rU)) were all purchased from Collaborative
Research.
B.
Binding protein
The E_. coli DNA-binding protein was purified as described .
[ H]-leucine-labeled protein (specific activity: 200 cpm/yg)
was isolated from a leucine auxotroph grown in minimal media
in the presence of [ H] leucine.
The concentration of binding
protein, where expressed in molarities, refers to the concentration of the monomer, assuming a molecular weight of 22,000.
C.
Fluorescence measurements
Fluorescence measurements were made on a MPF-3 PerkinElmer fluorescence spectrophotometer using an excitation wavelength of 285 nm and an emission wavelength of 350 nm.
Measure-
ments were taken at 5°C in 0.02 M Tris-HCl pH 7.4; all solutions were sterilized by Millipore (0.22 p pore size)
filtration to remove particular matter.
D.
Nucleotide concentrations
All nucleotide concentrations are expressed as the concentration of mononucleotides.
Concentrations were determined
spectrophotometrically, and the following extinction coefficients were employed with the nucleid acid dissolved in 0.02 M
Tris-HCl pH 7.4.
4
til
Nucleic acid
/•»
x l0
SS-DNA
12.0
Double-strand DNA
10.0
tRNA
10.0
T
Reference
poly(dA)
8.4
(5)
poly(dT)
8.1
(5)
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Nucleic acid
Reference
poly(dC)
6.6
(6)
poly(rC)
5.3
(7)
poly(rA)
9.6
(7)
poly(rU)
9.6
(7)
Oligodeoxythymidylic
acids
S.I
E.
Other techniques
Protein concentrations were determined by the method of
Q
Lowry et_ a_l_-
Sedimentation analysis of binding protein com-
plexes were performed using a linear glycerol gradient (10-30%)
in 0.01 M Tris-HCl pH 7.6, 10' 4 M EDTA.
Centrifugation was at
5°C at 150,000 x g, and the times of sedimentation are given in
the figure legends.
Fractions were collected from the bottom and
counted for radioactivity.
Values of the apparent K,.
were determined as follows: the
fluorescence from protein was measured.
Then nucleic acid was
added and after 5 minutes, the value of the fluorescence recorded.
Additional nucleic acid was added and the fluorescence recorded
again.
This was continued until no further decrease in fluores-
cence was seen following nucleic acid addition.
That fluorescence
was at a minimum aft°r the final addition was shown by adding
single-stranded DNA at high concentrations and showing that no
further decrease in fluorescence was obtained.
K,.
The apparent
was calculated from the curve (S0% relative fluorescence
quench) of fluorescence vs. nucleic acid concentration.
value for K,.
Each
was the average of four determinations performed
at 13 nM and 26 nM protein.
RESULTS
A.
Fluorescence measurements
UV radiation maximally excites the protein at 285 nm giving
rise to a fluorescence emission whose maximum is at 345 nm
(Figure 1). In the presence of increasing concentrations of
SS-DNA, the fluorescence emission is quenched by an amount
directly proportional to the concentration of DNA added (at low
concentrations).
When the binding protein is totally complexed
with DNA, only two-thirds of the initial fluorescence is maximally
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300
Figure 1.
350
Wovelength
400
( nm)
Fluorescence emission spectra of binding protein in
the presence of single-strand DNA.
(a)
Binding protein 0.1 yM; (b) +0.18 yM DNA; (c) +0.51
VlM DNA; (d) +0.66 uM DNA; (e) no protein + 0.66 yM DNA.
quenched.
Larger amounts of DNA failed to further quench the
fluorescence.
The fluorescence spectrum of pure binding protein
is similar to that exhibited by tryptophan, and the intensity
of the emission is directly proportional to protein concentration over at least the 20-fold range used in this report.
The
information contained in Figure 1 can be expressed such that the
relative fluorescence, at a particular emission wavelength, is
a function of the input nucleotide concentrations.
A typical plot
of the relative fluorescence versus nucleotide concentrations of
different nucleic acids is shown in Figure 2.
Poly(dT) exhibits
the greatest quenching effect; it is two-fold better than SS-DNA
at the same concentration.
SS-DNA, however, quenched the fluores-
cence due to binding protein 1000 times more efficiently than
double-stranded DNA.
This value represents the minimum difference
in apparent affinity between single- and double-strand DNA and
may only reflect 0.1% contamination of SS-DNA in the doublestrand DNA preparation, a conclusion supported by the observation that passage of the phenol extracted native DNA through nitrocellulose, under conditions where SS-DNA
is adsorbed, reduced
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100
2
30
I
Nucleotide Concentration, ^ M
Figure 2.
Quenching of binding protein fluorescence by various
nucleic acids
Fluorescence of binding protein (130 nM) alone is arbitrarily
set at 100% after correction for apparent fluorescence
caused by light scattering. Maximal absorption of light at
the excitation wavelength by added DNA was always less than
5%, since final DNA concentrations were, at most, 3 uM.
This change in intensity of emission due to added nucleic
acid was rapid, occurring faster than measurements could
be taken. Measurements of relative fluorescence were,
however, taken over a period of time to ensure true equilibrium had been attained. Figure 2a: • - double-stranded
DNA;B - single-stranded DNA; A-poly (dT) ; A-(pT).,;
°-(pT) 8 ; Figure 2b: O-tRNA; »-poly (rU).
the apparent K,.
by an order of magnitude (data not shown).
An unexpected result was obtained using the defined-length
oligonucleotides (pT) g and (pT) 1 6 .
As is shown in Figure 2a,
there is a dramatic decrease in the ability of the oligonucleotide to quench binding protein fluorescence where the chain
length is reduced from 16 to 8 nucleotides.
As is shown below,
this decrease reflects a large increase in the apparent
K
J^SS-
Little difference was noted, relative to (pT) g in the apparent
binding of the oligonucleotides (pT) 4 >
(pT) 6 , or (pT) 1 Q (data
not shown).
Binding protein, in addition to binding to DNA, exhibits
a lack of specificity in that poly(rU) quenches the fluorescence
of the protein with an efficiency about half that of poly(dT).
On the other hand, tRNA, which has considerable secondary structure,
still interacts with binding protein but with a reduced affinity
(relative to poly(rU)).
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B.
•Stoichiometry of binding and equilibrium constants for
dissociation by fluorescence spectroscopy
It is apparent from the data shown in Figure 2 and also from
the fact that binding protein only dissociated from denatured DNAcellulose in 2 M NaCl that the affinity of the protein for SS-DNA
is very high; i. e., that in the presence of excess binding
protein, SS-DNA is predominantly complexed to the protein; only
a small fraction remains free in solution.
At high protein con-
centration (130 nM), therefore, the equation:
[DNA] f r e e
can be simplified by assuming that [DNA]fre
«
[DNA]
.
,
yielding the equation:
t DNA Unput = ^ c o m p l e x
=
C s " i c h i °» e «rt x [BP] c o n l p l e x .
Thus, under these conditions, the amount of input DNA required
to fractionally saturate the binding protein present is a direct
measurement of the stoichiometry between binding protein and DNA.
From the data shown in Figure 2, this stoichiometry may be calculated to show that one binding protein monomer binds to 7-8
nucleotides of either DNA (2a) or RNA (2b). Having determined
the stoichiometry of binding, the DNA concentrations are henceforth expressed in terms of non-overlapping binding sites, which
is equal to total nucleotides divided by 8.
In order to calculate the K,.
of the complex, the initial
protein concentration was reduced to 13 nM.
At this concentra-
tion, equation (1) above cannot be simplified to [DNA].
=
[DNA]
, . For the reaction:
1
'complex
DNA-Protein complex •*• DNAv
*•
+ Protein,
free
free
The Law of Mass Action gives:
diss ~
[Protein]
[DNA - Protein
complex]
free
At one-half saturation of the protein with DNA, where [Protein]£ree=
[DNA-protein complex], K,.
tion.
is given by the free DNA concentra-
The values for the apparent K J - S S > calculated in the above
manner as described in Materials and Methods, are given in Table 1.
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Table 1. Binding constants of various nucleic acids to binding
protein measured by fluorescence quenching
Apparent equilibrium
dissociation constant(nanomolar)*
Nucleic <icid
poly(dT)
1
0 8
±
8
poly(dA)
1.4 + 0
single-strand DNA
2.0 + 1 3
(pT) 1 6
2.3 + 0
poly(rU)
1.9 ±
8
0 8
poly(dC)
15
±
1
poly(rA)
33
±
8
tRNA
poly(rC)
CpTJg
double-strand DNA
43
+
8
120
+
8
430
± 13
2000
poly(dT) in 0 32 M NaCl
40
poly(rU) in 0 32 M NaCl
200
*These values are all calculated assuming that one binding
protein monomer complexes with eight nucleotides. Furthermore,
they represent the binding of the monomeric form of the protein
to nucleic acid. It has also been assumed, in the case of the
naturally occurring nucleic acids, that the protein has an equal
affinity for all octanucleotides, regardless of base composition
and molecular conformation. We have not assumed overlapping
binding sites as discussed by McGhee and von Hippel , because
of the uncertainty of the number of binding sites assignable to
short oligo nucleotides, i. e., end effects. This treatment of
the data does not significantly alter the relative magnitude
of the changes in binding constants between oligomer and polymer.
These constants are valid only for the specific conditions
employed and may be altered considerably by changes in pH, temperature, or ionic strength.
the apparent K,.
The effect of ionic strength on
is especially large; the addition of NaCl to
a final concentration of 0.32 M to the standard buffer increases
the apparent K d i s s by a factor of 40 for poly(dT) and of 100 for
poly(rU).
Under the conditions employed, SS-DNA and the homo-
polymers poly(dA), poly(dT), and polyCrll) have all comparbale
binding of 10" 9 moles liter'1.
This result and the fact that
both poly(dT) and poly(rU) exhibit little secondary structure in
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solution (which might otherwise affect the K,.
) clearly shows
a lack of absolute specificity of the binding protein for the
sugar moiety of nucleic acid.
The polyribonucleotides examined, other than poly(rU), show
a decreased affinity for binding protein relative to their deoxyribonucleotide counterparts, and may reflect the affinity of the
protein for various secondary structures of the nucleic acids in
solution.
Poly(dA), which is known to have secondary structure in
solution , is still bound by binding protein with a high affinity.
The DNA from bacteriophage fd is also bound with high affinity.
It
is a circular single-stranded molecule which has no extensive
regions of ordered base-paired structure; only 1% of the DNA is
q
resistant to single-strand-specific nucleases .
Binding protein
does not bind to poly(dC) as tightly as the other deoxyhomopolymers
tested.
It is not clear whether this decrease in affinity is due
to inherent base specificity of binding protein or is due to the
secondary structure of this nucleic acid.
Binding protein binds to tRNA with a K d i
approximately equal
to that of poly(rA), and yet no allowance has been made in our
calculations for the fact that 40-50% of the nucleotides in tRNA
are in double-strand form, nor that the unpaired regions are not
eight nucleotides in length (i. e., on binding sites of a binding
protein monomer).
As the binding site of the monomeric form of the binding
protein is eight nucleotides, a simplistic viewpoint is that complex formation with (pT)fi represents the binding of a monomer to
a free DNA molecule, whereas complex formation with (pT) 1 6 represents the binding of two monomers adjacent to each other; i. e.,
on contiguous binding sites.
The major difference in the binding
constants of the two oligomers (Table 1) therefore reflects the
cooperative interaction of protein molecules on DNA.
Further-
more, there is no significant increase in affinity for nucleic
acids longer than (pT), 6 in that poly(T) and DNA have binding
constants indistinguishable from that of (pT).,.*
•Our inability to observe a difference in the binding of
(pT)l6 and poly(dT), which might be expected on the basis of
cooperative binding events for the polymer versus only one for
the oligomer may reflect the lack of sensitivity of our method for
the determination of binding constants lower than that of (pT)j^,
since under the conditions of the measurements, about 90% of the
nucleic acid is bound and 10% is free.
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C.
Stoichiometry of binding and rate constant of dissociation by sedimentation analysis
Since the apparent K^
values were obtained using stoichio-
metry of protein: nucleotide as 1:8, a ratio calculated from fluorescence data,
the stoichiometry of binding was also examined
by sedimentation of a protein - DNA complex for independent confirmation.
Varying amounts of DNA were mixed with a constant amount
of [ H]-labeled binding protein and the mixture was then layered
on top of a 10-30% (v/v)
glycerol gradient and centrifuged such
that the DNA-protein complex would sediment 85% of the way through
the gradient.
Assuming that all the radioactivity in the top 15%
of the gradient represents protein that was not bound to DNA
before sedimentation, then the remainder (that which has sedimented)
is directly proportional to the amount of protein that was bound
to DNA initially.
The values for the amount of bound protein, ob-
served as a function of input DNA, are shown in Figure 3.
There
is a linear relationship between the weight of input DNA and
the weight of protein bound up to a ratio of 1:8.5 (DNA: protein).
On a molecular basis, this represents the binding of one protein
monomer (M. W. 22,000) to eight nucleotides (M. W. 2,600), a
value which is in agreement with that obtained by fluorescence
measurements and also confirms the original stoichiometry calculated by Sigal et_ a_l_. .
As the DNA-protein complex sediments through the gradient,
it dissociated to give free protein and free DNA.
The sedimenta-
tion properties of fd DNA and an fd-DNA binding protein complex
are similar,
and both species will sediment faster than the disso-
ciated free protein.
It is assumed for the purposes of calculation
that a protein molecule, once dissociated, is unable to re-associate
with DNA, and one can therefore study the dissociation process
as an irreversible reaction.
Protein which sedimented to or
beyond a given position in the gradient is considered to be
that amount of protein still bound at the time when the complex
reached that position in the gradient.
Centrifugation was for
six hours and thirty fractions were collected from the gradient.
We assume that each fraction represents twelve minutes on a time
scale, and therefore, one can calculate the amount of bound protein as a function of time.
1830
As is shown in Figure 4, a plot of
Nucleic Acids Research
^o
Figure 3.
Stoichiometry of DNA-protein complexes
3.
H-binding protein (15 pg) was mixed with various weights
of fd DNA at 0°C. The mixture was then sediraented through
a linear (10-30% v/v) glycerol gradient for 6 hours at
150,000 x g. The weight of protein that sedimented more
than 5 fractions and therefore at equilibrium (before centrifugation) had been bound to DNA is plotted as a function
of the weight of DNA added.
Figure 4.
Dissociation of DNA protein complexes as a function of
time
Conditions for the reaction mixtures and the glycerol
gradients were as described in Figure 3. Other details
are given in the text. Calculations are made from the
slopes obtained with data between 1 and 4 hours. The
curves represent (top to bottom) data obtained with 1.0,
1.2, 1.5, and 1.7 ug of input DNA.
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the natural logarithm of the percentage of the initially bound
protein that is still bound as a function of time results in a
linear relationship (ignoring the first five fractions where free
binding protein is present).
The linearity is independent of
the concentration of input DNA indicating that the dissociation
process is a first-order reaction.
The lack of dependence on
concentration suggests that dissociation is irreversible.
The
slope of the derived plot is equal to -k ,,.. . The dissociation
rate constant (k „ ) thus calculated is approximately 4 x 10"
sec" , and the half-life of the complex is in the order of 300
minutes. The rate constant for complex formation is therefore
4
1 1
2 x 10 liter mole
sec . The value of the k „ is a minimal
value since we assumed that dissociation during centrifugation
is irreversible.
This method has been used previously to esti-
mate k . f for the T7-induced DNA binding protein.
D.
Sedimentation of binding protein-oligonucleotide complexes
It was stated above that binding protein bound to DNA initially as a monomer, even though the isolated form of the protein
is tetrameric.
It may be argued that the apparent K ^
for the
oligonucleotides is a measure of the ability of the protein to
"line-up" the oligomers within the tetrameric protein structure
and the basic binding unit of the protein is in fact the tetramer.
This possibility was excluded and the subunit nature of binding
was confirmed by sedimentation of a (pT)^,-binding protein complex.
As is seen in Figure 5, the sedimentation profile of the (pT) 1 6 binding protein complex is slower than that of binding protein
alone, and, therefore, much slower than would be predicted for a
complex of the tetrameric form of the protein complexed to two
molecules of (pT) 1 6 -
The observed complex sediments with an
apparent mass of 54,000 daltons.
Thus, the active binding species
of the protein is either the dimeric or monomeric form.
These
two possibilities could not be further distinguished by the
sedimentation of oligomers smaller than CpT),,, since these complexes were not stable to centrifugation.
DISCUSSION
The E_. coli DNA-binding protein has been shown to bind tightly
and cooperatively to single-strand nucleic acid.
The protein has
a slightly greater affinity for the homopolymers poly(dA) and
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Ol
0
0
Figure 5.
10
20
Fraction Number
Sedimentation of an oligonucleotide-protein complex
H-binding protein (45 yg) was mixed with 5'-[ P]-(pT)
16
(2.8 yg) and sedimented through a linear (10-30% v/v)
glycerol gradient (see Methods) for 24 hours at 230,000
x g. Fractions were collected and counted directly for
[•^H] and [32p] radioactivity. • — • : binding protein;
0 — 0 : (pT)j^. The dashed line represents the sedimentation of (pT)}6 in the absence of binding protein and the
arrow marks the sedimentation position of the tetrameric
form of binding protein in the absence of any added DNA.
poly(dT) than for poly(dC) or for SS-DNA.
The protein therefore
appears to exhibit a small degree of base specificity in binding.
The protein also binds to RNA; in fact, the binding to poly(rU)
is as tight as to SS-DNA and only slightly less than to poly(dT),
though the other ribohomopolymers bind with much less affinity
than do their deoxy- counterparts.
This probably reflects an
increased affinity of the protein for deoxy- rather than ribonucleotides, but may also be a function of the different secondary
structures of the various homopolymers.
The failure to detect
binding of the protein to bacteriophage R17 RNA1 may be due to
the large degree of secondary structure exhibited by viral RNAs,
or the difference in binding conditions.
The protein binds to double-strand DNA at least three orders
of magnitude weaker than it does to single-strand DNA.
Under
the same experimental conditions, the protein bound to tRNA with
remarkably high affinity.
It is possible that in the tRNA mole-
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cule, the looped-out regions are sufficiently exposed to allow
the first protein molecule to bind, and, because of the increased
affinity of a second protein molecule for a contiguous site, the
protein may then tend to cover the entire molecule, resulting in
denaturation of the tRNA.
This statement implies that binding
protein alone is not able to denature native DNA when the latter
is in a perfect duplex structure, but requires a localized nonbase-paired region in order to form an initial complex of proteinnucleic acid.
Once this initial complex is formed, then the
increased binding affinity of the second and subsequent protein
molecules may catalyze the separation of the DNA strands.
This
is in full agreement with the observation, based on partial
denaturation mapping of A DNA in that the protein expands rather
than initiates denaturation loops.
By the use of oligonucleotides of defined length, we have
measured directly the degree of cooperativity exhibited by the
protein and found that whereas the binding of an isolated protein
molecule (binding to (pT)g) has an apparent K,.
of 4 x 10"
M,
the binding of two protein molecules on contiguous sites binding
Q
to (pT) 1 6 has an apparent dissociation constant of 2 x 10
M.
This cooperative binding is a feature common to all the DNAbinding proteins characterized to date, in particular, for the
T7-induced protein
, the fd-gene-5 protein
'
, and the T4 gene-
32 protein . Only for the latter two proteins has an estimate
of the cooperativity been made: a factor of about 80 for the
gene-32 protein and about 60 for the gene-5 protein. The extent
of cooperativity observed in this study cannot be accurately determined but can be approximated to a minimum of 50. The latter
figure derives from considering the overlapping binding site
14
treatment of McGhee and von Hippel.
The DNA-binding proteins
thus possess an approximately constant degree of cooperativity,
and their mechanism for binding to DNA may all be comparable.
Cooperativity is likely to be an important feature of the DNAbinding proteins since it allows the proteins to saturate fully
and thus fully protect single-strand DNA regions from enzyme
action or fully promote enzyme action.
A further common property is the apparent lack of complete
nucleotide specificity resident in the DNA-binding proteins, but
since they all appear to preferentially bind to A-T rich regions
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Nucleic Acids Research
in DNA, it may be argued that, under conditions where binding is
limited, those bases are the only ones bound.
We have shown that
the apparent K d i s s of the £. coli protein to the homopolymers
poly(dA) and poly(dT) is lower than for poly(dC) or SS-DNA, and
it is probable that a similar result would be obtained for the
other DNA-binding proteins.
The weaker binding to contiguous
dC residues may have physiological significance.
Both the E_. coli and T4 proteins have apparent dissociation
-9
-1
constants to DNA of approximately 10
moles liter
, which
signifies a tighter binding than exhibited by most nucleic
acid enzymes for SS-DNA, though the binding is very much weaker
than that observed for the lac or X repressors to their operators.
However, these latter proteins bind to highly specific nucleotide
sequences, different in nature from the non-specific binding
exhibited by the DNA-binding proteins.
The precise physiological role of any of these proteins is
still unclear.
The T4-gene-32 protein is apparently involved in
all three known DNA synthetic processes; i. e. , replication,
recombination, and repair,
'
'
while the fd gene-5 protein
probably serves some regulatory function in preventing progeny
SS-DNA from being converted to a double-strand replicative form.
'
The T7 and E_. coli proteins are not characterized as well since
no mutants in the genes coding for these proteins are yet known.
However, it would seem that, at least in the T7 system where there
are 10 T7 binding protein molecules/cell, "naked" SS-DNA as
11
such does not exist in_ vivo, but is, as was originally suggested,
always complexed with the T7 DNA-binding protein.
There are
about 400 molecules/bacterium of the E_. coli protein, sufficient
to cover about 3000 nucleotides.
Whatever process this DNA may
be participating in, be it replication, recombination, or repair,
it seems clear that any other enzymes involved in catalyzing
this hypothetical process must be able to function in the presence
of binding protein.
For example, the protein inhibits DNA poly-
merase I, but stimulates DNA polymerase II ; it is therefore
apparent that the latter enzyme and binding protein function
together in some common process.
Conversely, in an in vitro
DNA polymerase I-catalyzed reaction, binding protein must be prevented from binding at the template site, either by a control
mechanism or by allowing no SS-DNA in the process.
In addition,
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Nucleic Acids Research
since the protein inhibits the single-strand but not the doublestrand activities of exonuclease V, the recB-C nuclease (S. Linn
and V. MacKay, personal communication) binding protein may serve
as a regulatory process in protecting SS-DNA from degradation.
There is a definite need for the characterization of an E_. coli
strain with an altered DNA-binding protein such that its biological function can be determined.
ACKNOWLEDGEMENTS
We would like to thank Drs. M. Fox, P. Sharp, and C. Walsh
of the Department of Biology at MIT for their help in critically
evaluating this manuscript.
This work was supported in part by
grant No. B36649 from the National Science Foundation and in part
by grant No. R-R01-GM20363-02 from the National Institutes of
Health.
"T'resent address: Imperial Cancer Research Fund, Burtonhole Road,
London N W 7 IAD, England
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