Escherichia coli Replicative Helicase PriA
Protein-Single-stranded DNA Complex
STOICHIOMETRIES, FREE ENERGY OF BINDING, AND
COOPERATIVITIES*
Maria J.
Jezewska,
Surendran
Rajendran, and
Wlodzimierz
Bujalowski
From the Department of Human Biological Chemistry and Genetics, the
University of Texas Medical Branch, Galveston, Texas 77555-1053
Received for publication, May 15, 2000, and in revised form, June 23, 2000
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ABSTRACT |
Analyses of interactions of the Escherichia
coli replicative helicase, PriA protein, with a single-stranded
(ss) DNA have been performed, using the quantitative fluorescence
titration technique. The stoichiometry of the PriA
helicase·ssDNA complex has been examined in binding
experiments with a series of ssDNA oligomers. The total site-size of
the PriA·ssDNA complex, i.e. the maximum number of
nucleotide residues occluded by the PriA helicase in the complex, is
20 ± 3 residues per protein monomer. However, the protein can
efficiently form a complex with a minimum of 8 nucleotides. Thus, the
enzyme has a strong ssDNA-binding site that engages in direct
interactions with a significantly smaller number of nucleotides than
the total site-size. The ssDNA-binding site is located in the center of
the enzyme molecule, with the protein matrix protruding over a distance
of ~6 nucleotides on both sides of the binding site. The analysis of
the binding of two PriA molecules to long oligomers was performed using
statistical thermodynamic models that take into account the overlap of
potential binding sites, cooperative interactions, and the
protein·ssDNA complexes with different stoichiometries. The intrinsic
affinity depends little upon the length of the ssDNA. Moreover, the
binding is accompanied by weak cooperative interactions.
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INTRODUCTION |
In the process of priming a DNA strand, the primosome, a multiple
protein complex is formed, which can translocate along the DNA while
synthesizing short oligoribonucleotide primers that are used to
initiate synthesis of the complementary strand (1-3). The PriA protein
is a key replication protein in Escherichia coli that plays
a fundamental role in the ordered assembly of the primosome. Originally, the protein was discovered as an essential factor during
the synthesis of the complementary DNA strand of phage 
X174 DNA (4,
5).
In vitro studies demonstrated that the PriA protein displays
multiple activities as follows: 1) specific binding of ATPs and dATPs
(4-8); 2) ATPase and dATPase activities strongly stimulated by a
specific DNA fragment, the primosome assembly site
(PAS)1 (7-8); 3) specific
strong binding to the PAS sequence (7-9); 4) nonspecific binding to
the ssDNA (7-10); and 5) 3' 
5' helicase activity specifically
stimulated by PAS (11, 12). These multiple activities reflect complex
interactions of the enzyme with different ingredients in the primosome,
including protein-protein and protein-DNA interactions (3, 12).
The gene encoding the PriA protein has been cloned and sequenced and
that of the encoded protein has been determined (13, 14). Current
biochemical data indicate that the native protein is a monomer, with a
molecular mass of 81.7 kDa, which is proposed to be the
predominant form of the protein in solution (7, 9).
In vivo functions of the PriA helicase are related to the
ability of the enzyme to interact with both ss- and dsDNAs (3, 7-10,
15, 16). Although the importance of understanding the PriA protein
interactions with a nucleic acid has been recognized, little is known
about the quantitative aspects of these interactions. Nothing is known
about the stoichiometry, i.e. the total site-size of the
PriA·ssDNA complexes with different ss- and dsDNAs, base specificity,
effect of solution conditions on the intrinsic affinity, the
cooperativity of the binding process, and the structure of the
complexes. Knowledge of the stoichiometry and structure of the
helicase-nucleic acid complex and the mechanism of binding is a
prerequisite for formulating any model of the mechanism of enzyme
functioning in DNA replication (17, 18). This includes enzyme
translocation on the DNA, catalysis of DNA unwinding, as well as the
functioning of the PriA helicase in the formation and translocation of
the primosome (19-20).
In this communication, we report, for the first time, quantitative
analyses of the interactions of the PriA helicase with a ssDNA, using
the quantitative fluorescence titration technique. We present evidence
that the helicase binds the ssDNA as a monomer with the total site-size
of the protein·ssDNA complex n = 20 ± 3 nucleotide residues. However, studies with a series of short oligomers
indicate that the ssDNA-binding site on the enzyme, directly engaged in
interactions with the nucleic acid, encompasses only 8 ± 1 nucleotide residues, significantly less than the total site-size of the
complex. The obtained results indicate that the enzyme has a single
ssDNA-binding site, located in the central part of the protein
molecule, with the protein matrix protruding over a distance of 6 ± 1 nucleotide residues on both sides of the ssDNA-binding site. The
data show the binding of PriA to the ssDNA is not accompanied by any
significant cooperative interactions.
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EXPERIMENTAL PROCEDURES |
Reagents and Buffers--
All solutions were made with distilled
and deionized >18 megohms (Milli-Q Plus) water. All chemicals were
reagent grade. Buffer C is 10 mM sodium cacodylate adjusted
to pH 7.0 with HCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 25% glycerol. The temperatures and concentrations of
salts in the buffer are indicated in the text.
PriA Protein--
Plasmid pET3c, harboring the gene of the
E. coli PriA protein, was a generous gift from Dr. K. Marians (Sloan Kettering). However, to obtain better overproduction,
the priA gene was placed in pET30a plasmid (Novagen).
The protein was purified using the procedure previously described (13).
The protein was >97% pure as judged by polyacrylamide electrophoresis
with Coomassie Brilliant Blue staining. The concentration of the PriA
protein was spectrophotometrically determined, with the extinction
coefficient 
280 = 1.06 × 105
cm
1 M1
(monomer) determined using an approach based on the Edeldoch method
(21-26).
Nucleic Acids--
Oligomers, dA(pA)5,
dA(pA)7, dA(pA)9, dA(pA)13,
dA(pA)15, dA(pA)17, dA(pA)19,
dA(pA)23, dA(pA)25, dA(pA)29,
dA(pA)34, dA(pA)39, and homopolymer, poly(dA)
were purchased from Midland Certified Reagents (Midland, Texas) and
Sigma. Oligomers were at least >95%, as judged by autoradiography on
polyacrylamide gels. The etheno derivatives of the nucleic acids were
obtained by modification with chloroacetaldehyde (19, 27). This
modification goes to completion and provides fluorescent derivatives of
the nucleic acid. The concentration of the etheno derivative of the
nucleic acids was determined using the extinction coefficient 3700 M1 cm1
(nucleotide) at 257 nm (28-32).
Fluorescence Measurements--
All steady-state fluorescence
titrations were performed using SLM-Aminco 48000S or 8100 spectrofluorometers. In order to avoid possible artifacts, due to the
fluorescence anisotropy of the sample, polarizers were placed in
excitation and emission channels and set at 90 and 55° (magic angle),
respectively (28-35). The binding was followed by monitoring the
fluorescence of the etheno derivatives of the nucleic acids
(
ex = 325 nm, em = 410 nm). Numerical
fits were performed using KaleidaGraph (Synergy, PA) and Mathematica
(Wolfram, IL).
The relative fluorescence increase of the nucleic acid,
F, upon binding the PriA protein is defined by Equation 1,
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(Eq. 1)
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where Fi is the fluorescence of the nucleic
acid solution at a given titration point "i," and
Fo is the initial value of the fluorescence of the
same solution. Fluorescence emission spectra have been corrected for
the instrumental factors using the software provided by the manufacturer.
Determination of Thermodynamically Rigorous Binding Isotherms of
the PriA Helicase·ssDNA Complexes--
In this work, we followed the
binding of the PriA protein to ssDNA oligomers, by monitoring the
fluorescence increase, Fobs, of their etheno
derivatives upon the complex formation. To obtain rigorous estimates of
the average degree of binding, 
i (number of the bound
protein molecules per ssDNA oligomer) and the free protein
concentration, PF, independent of any assumption about the
relationship between the observed spectroscopic signal and
i, we applied an approach previously described by us (19,
23-26, 29-32). Briefly, the experimentally observed Fobs has a contribution from each of the
different possible i complexes of the PriA helicase
with the ssDNA. Thus, the observed fluorescence increase is
functionally related to i by Equation 2,
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(Eq. 2)
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where Fi(max) is the molecular
parameter characterizing the maximum fluorescence increase of the
nucleic acid with the PriA protein bound in complex i. The
same value of Fobs, obtained at two different
total nucleic acid concentrations,
NT1 and
NT2, indicates the same physical
state of the nucleic acid, i.e. the degree of binding,
i, and the free PriA protein concentration, PF,
must be the same. The value of i and PF is then
related to the total protein concentrations, PT1
and PT2, and the total nucleic acid concentrations,
NT1 and NT2, at the same value
of Fobs, by Equations 3 and 4,
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(Eq. 3)
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(Eq. 4)
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where x = 1 or 2 (19, 29-32, 34, 35).
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RESULTS |
Determination of the Total Site-size of the PriA Helicase·ssDNA
Complex Using ssDNA Etheno Derivatives--
Binding of the PriA
helicase to the ssDNA homopolymers is not accompanied by an adequate
change in the helicase fluorescence to perform quantitative analyses of
the PriA-ssDNA interactions. However, we have found that formation of
the complex between the enzyme and the etheno derivative of the
adenosine polymer and oligomers causes a strong ~3-fold nucleic acid
fluorescence increase. Because at 325 nm (excitation wavelength) the
etheno adenine is predominantly excited, the observed fluorescence
increase must result from the quantum yield increase of the nucleic
acid. It should be noted that incorporation of A into the polymer
induces an 8-10-fold quenching of the A fluorescence, as compared
with the free mononucleotide (36, 37). Thus, the strong nucleic acid
fluorescence increase, when bound to the PriA helicase, indicates that
some of the quenching processes have been removed in the complex with
the helicase (see "Discussion").
The dramatic enhancement of the ssDNA etheno derivative fluorescence
provides an excellent signal to monitor the PriA-ssDNA interactions and
to perform the high resolution measurements of the stoichiometry and
mechanism of the helicase·ssDNA complex formation. However,
quantitative fluorescence titrations of the etheno derivative of
poly(dA) and poly(dA), with the PriA helicase, were hindered by the
fact that the protein-polymer nucleic acid complex precipitates at high
concentrations of the protein and the nucleic acid (data not shown).
Nevertheless, these data indicate that the total site-size of the
helicase·ssDNA complex, i.e. the maximum number of
nucleotides occluded by the enzyme in the complex, is lower than ~25
nucleotide residues. Therefore, to address the fundamental problem of
the stoichiometry of the helicase·ssDNA complex, we performed a
series of quantitative studies using several ssDNA oligomers with
different numbers of nucleotide residues.
Fluorescence titrations of the 24-mer, dA(pA)23, with
the PriA protein at two different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, are
shown in Fig. 1a. At higher
nucleic acid concentrations, a given relative fluorescence increase is
reached at higher PriA concentrations. This increase results from the
fact that, at higher DNA concentrations, more protein is required to
obtain the same degree of binding, i. The selected
nucleic acid concentrations provide separation of the binding isotherms
up to the relative fluorescence increase of ~2.3.
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Fig. 1.
a, fluorescence titrations of the
24-mer dA(pA)23 with the PriA protein
(ex = 325 nm, em = 410 nm) in buffer C
(pH 7.0, 10 °C), containing 100 MNaCl, at two different
nucleic acid concentrations:  , 4.7 × 107 M;  , 1.2 × 105 M (oligomer). The solid
lines are nonlinear least squares fits of the titration curves,
using a single-site binding isotherm (Equation 6) that takes into
account the fact that the ssDNA-binding site engages only 8 residues of
the nucleic acid in direct interactions. Intrinsic binding constant
Ki = 4.4 × 104
M1 and relative fluorescence
change Fmax = 2.8. b, dependence
of the relative fluorescence of the 24-mer, F, upon the
average number of bound PriA proteins (). The solid line
follows the experimental points and has no theoretical basis. The
dashed line is the extrapolation of F to the
maximum value of Fmax = 2.8.
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To obtain binding parameters, independent of any assumption about the
relationship between the observed signal and the degree of binding,
i, the fluorescence titration curves, shown in Fig.
1a, have been analyzed, using the approach outlined under "Experimental Procedures" (19, 23-26, 29-32). Fig. 1b
shows the dependence of the observed relative fluorescence increase of
the 24-mer as a function of the average degree of binding,
i, of the PriA helicase. Short extrapolation to the
maximum fluorescence change, Fmax = 2.8 ± 0.2, shows only one molecule of the PriA helicase binding to the
24-mer. The same 1:1 stoichiometry and the same affinities (see below)
were obtained with higher and lower 24-mer concentrations providing
direct thermodynamic evidence that the enzyme exists in our solution
conditions predominantly as a monomer, and the monomer is the form that
binds the ssDNA. This is in excellent agreement with early studies that
indicated that the PriA helicase exists in solution as a monomer (7, 9). Additionally, using the analytical ultracentrifugation technique,
we determined that the sedimentation coefficient of the enzyme,
s20,w = 5.9 ± 0.15, remains
constant, within experimental accuracy, over a large protein
concentration range (data not shown). These results indicate that the
PriA helicase exists as a monomer in the protein concentration range
studied in this work.
The maximum stoichiometry of the enzyme-ssDNA oligomer is different in
the case of the 30-mer, dA(pA)29, although this
oligomer is only six nucleotide residues longer than the 24-mer.
Fluorescence titrations of the dA(pA)29 with the PriA
helicase at two different nucleic acid concentrations, in buffer C (pH
7.0, 10 °C), containing 100 mM NaCl, are shown in Fig.
2a. Separation of the
titration curves allowed us to determine the average degree of binding, i, up to ~1.6, a value significantly higher than in the
case of the 24-mer. The dependence of the relative fluorescence increase of the 30-mer, as a function of the average degree of binding
of the PriA helicase on the oligomer, is shown in Fig. 2b.
Extrapolation to the maximum fluorescence increase
Fmax = 2.5 ± 0.2 provides
i = 2.2 ± 0.2. Thus, the presence of an extra six
nucleotide residues in the 30-mer, as compared with the 24-mer,
provides enough interaction space for the binding of the second PriA
protein molecule.
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Fig. 2.
a, fluorescence titrations of the 30-mer
dA(pA)29 with the PriA protein (ex = 325 nm, em = 410 nm) in buffer C (pH 7.0, 10 °C),
containing 100 mM NaCl, at two different nucleic acid
concentrations: , 4.7 × 107
M; , 2.1 × 106
M. The solid lines are nonlinear least squares
fits of the titration curves, using the statistical thermodynamic model
for the binding of two PriA molecules to the 30-mer, described by
Equations 7, 10, and 13. The intrinsic binding constant
Ki = 1.3 × 105
M1, cooperativity parameter
 = 11, and relative fluorescence change
F1 = 1.25, and Fmax = 2.5. b, dependence of the relative fluorescence of the
30-mer, F, upon the average number of bound PriA proteins
(). The solid line follows the experimental points and
has no theoretical basis. The dashed line is the
extrapolation of F to the maximum value of
Fmax = 2.5.
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However, the stoichiometry of the two PriA molecules bound per ssDNA
oligomer is not changed when the length of the ssDNA oligomer is
further increased by an additional 10 residues. Fluorescence titrations
of the 40-mer, dA(pA)39, with the PriA helicase at two different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, are shown in Fig.
3a. Separation of the
titration curves allowed us to determine the average degree of binding,
i, up to ~1.5. The titration at high nucleic acid
concentrations could not reach a plateau because of the precipitation of the protein-nucleic acid complex at high protein and nucleic acid
concentrations for this longest ssDNA oligomer studied. The dependence
of the relative fluorescence increase of the 40-mer, as a function of
the average degree of binding of the PriA helicase on the oligomer, is
shown in Fig. 3b. Extrapolation to the maximum fluorescence
increase Fmax = 3.5 ± 0.2 provides
i = 2 ± 0.2.
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Fig. 3.
a, fluorescence titrations of the 40-mer
dA(pA)39 with the PriA protein (ex = 325 nm, em = 410 nm) in buffer C (pH 7.0, 10 °C),
containing 100 mM NaCl, at two different nucleic acid
concentrations: , 4.6 × 107
M; , 4 × 106
M. The solid lines are nonlinear least squares
fits of the titration curves, using the statistical thermodynamic model
for the binding of two PriA molecules to the 40-mer, described by
Equations 9, 12, and 15. The intrinsic binding constant
Ki = 6 × 104
M1, cooperativity parameter
= 0.8, and relative fluorescence change
F1 = 1.7, and Fmax = 3.5. b, dependence of the relative fluorescence of the
40-mer, F, upon the average number of bound PriA proteins
(n). The solid line follows the experimental
points and has no theoretical basis. The dashed line is the
extrapolation of F to the maximum value of
Fmax = 3.5.
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Quantitative analysis of the maximum stoichiometry of PriA·ssDNA
complexes has been performed for a series of ssDNA oligomers. The
dependence of the maximum number of bound PriA molecules per ssDNA
oligomer, determined using the approach (see "Experimental Procedures") upon the length of the oligomer, is shown in Fig. 4. Oligomers from 8 to 26 nucleotide
residues bind a single PriA molecule. Transition from a single enzyme
bound per oligomer to two bound protein molecules per ssDNA occurs
between 26- and 30-mers (Fig. 4). However, a further increase in the
length of the oligomer, up to 40 residues, does not lead to an
increased number of bound PriA molecules per ssDNA oligomer. These data
provide the first indication that a total site-size of the PriA·ssDNA
complex is less than 24 nucleotide residues, but it must contain at
least 15 nucleotide residues per bound protein molecule (see below).
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Fig. 4.
The maximum number of bound PriA molecules,
as a function of the length of the ssDNA oligomer in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl. The solid
lines follow the experimental points and have no theoretical
bases. The number of the bound PriA molecules has been determined using
the quantitative approach outlined under "Experimental
Procedures."
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Number of Nucleotide Residues Directly Engaged in Interactions with
the ssDNA-Binding Site of the PriA Helicase--
The total site-size
of a large protein ligand-DNA complex corresponds to the DNA fragment,
which includes nucleotide residues directly involved in interactions
with the protein, its DNA-binding site, and the nucleotides not engaged
in direct interactions (38-40). The latter are prevented from
interacting with another protein molecule by the protruding protein
matrix of the previously bound protein molecule over nucleotides
adjacent to the binding site (37-39). This is clearly evident in the
case of the PriA complexes with 8- and 26-mers (Figs. 3 and 4). Both
oligomers can bind only a single enzyme molecule, yet the length of the
26-mer is more than three times longer than the length of the
8-mer.
An accurate estimate of the site-size of the PriA helicase·ssDNA
complex, using ssDNA oligomers, requires the evaluation of the number
of nucleotide residues directly engaged in interactions with the
ssDNA-binding site of the helicase. Fluorescence titrations of the
dA(pA)13, dA(pA)9,
dA(pA)7, and dA(pA)5 with the PriA
helicase in buffer C (pH 7.0, 10 °C), containing 100 mM
NaCl, are shown in Fig. 5. Titration
curves of the 14-, 10-, and 8-mers show similar fluorescence increases,
although slightly decreasing as the length of the nucleic acid
decreases. Recall, a single molecule of the PriA helicase, binds to
14-, 10-, and 8-mers (Fig. 4). Moreover, the binding to these oligomers
is characterized by very similar intrinsic affinities (Table
I). A very dramatic drop in the affinity
and the accompanying fluorescence increase occurs when the number of
nucleotide residues is lower than eight, with the affinity of the 6-mer
being beyond the quantitative analysis in our solution conditions (K
1 × 103
M1). These data clearly indicate
that the number of nucleotide residues directly involved in
interactions with the ssDNA-binding site of the PriA helicase, within
the total site-size of the protein·ssDNA complex, is 8 ± 1. Moreover, the fact that the helicase binds only a single 8-mer molecule
indicates that the enzyme possesses only one strong ssDNA-binding
site.
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Fig. 5.
Fluorescence titrations of the
dA(pA)13,
dA(pA)9,
dA(pA)7,
and
dA(pA)5
with the PriA helicase (ex = 325 nm, em = 410 nm) in
buffer C (pH 7.0, 10 °C), containing 100 mM NaCl.
, dA(pA)13; , dA(pA)9;  ,
dA(pA)7;  , dA(pA)5.
Concentrations of 14-, 10-, 8-, and 6-mer are 4.5 × 107 M (oligomer). The solid
lines are nonlinear least squares fits using a single-site binding
isotherm (Equation 6) that takes into account the fact that the
ssDNA-binding site engages only 8 nucleic acid residues in direct
interactions. Intrinsic binding constants and maximum fluorescence
increases accompanying the complex formation are included in Table
I.
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Table I
Thermodynamic and spectroscopic parameters characterizing the binding
of the E. coli PriA helicase to ssDNA oligomers which binds only one
enzyme nucleotide in buffer C (pH 7.0, 10 °C) containing 100 mM NaCl
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The Total Site-size of the PriA Helicase·ssDNA Complex, Model of
PriA Protein-ssDNA Interactions--
The transition from a single PriA
molecule bound per ssDNA oligomer to two molecules bound per oligomer
occurs between 26- and 30-mers, suggesting that the minimum, total
site-size of the PriA·ssDNA complex is 15 nucleotide residues per
bound protein (Figs. 2 and 4). On the other hand, binding studies with
short oligomers indicate that the protein engages only 8 nucleotide residues in interactions with its ssDNA-binding site (see above). These
data strongly suggest that a significant part of the total site-size of
the complex (at least 7 residues) results from the protruding of the
large protein molecule over the residues adjacent to the ssDNA-binding site.
If the ssDNA-binding site of the protein is located on one side of the
molecule, with a part of the enzyme protruding over the extra 7 residues, then the 24-, 26-, and 40-mers would be able to accommodate
two and three PriA molecules, respectively (Fig.
6). This is not what is experimentally
observed. One PriA molecule binds to the 24- and 26-mers, and only two
enzyme molecules bind to the 40-mer (Fig. 4). Therefore, the model
presented in Fig. 6 cannot represent the PriA·ssDNA complex (see
"Discussion"). Next, we consider a model where the ssDNA-binding
site, which engages 8 nucleotide residues, is located in the central
part of the protein, as depicted in Fig.
7. The protein molecule now has two parts
that are protruding over 6 nucleotide residues on both sides of the
ssDNA-binding site. In this model, only one PriA molecule can bind to
the 24- and 26-mers. This is because the first bound molecule can now
block at least 14 nucleotide residues. For efficient binding, the
second protein molecule also needs a fragment of at least 14 nucleotide
residues, which is larger than the remaining 11 and 12 residues of the
24- and 26-mers, respectively. The two bound PriA molecules require at
least 28 nucleotide residues of the ssDNA. On the other hand, this
allows two molecules of the enzyme to bind to the 30-, 35-, and 40-mers (see Fig. 4). In the case of the 40-mer, the remaining fragment of 8 residues is 6 nucleotides too short, i.e. it does not
provide efficient interacting space to allow the third PriA protein to associate with the oligomer. This is exactly what is experimentally observed (Fig. 4). Therefore, the model of the protein-ssDNA, presented
in Fig. 7, adequately describes all experimentally determined stoichiometries of the PriA with the series of ssDNA oligomers examined
in this work (see "Discussion"). Moreover, these data and the
analyses indicate that the actual total site-size of the PriA·ssDNA
complex is 20 ± 3 nucleotide residues and include 8 residues
encompassed by the ssDNA-binding site of the enzyme, as well as ~12
residues occluded by the protruding protein matrix (Fig. 7).
Examination of the intrinsic affinities of the enzyme to different
ssDNA oligomers provides additional support for the proposed model of
the PriA·ssDNA complex (see below).
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Fig. 6.
Schematic model for the binding of the PriA
helicase to the ssDNA, based on the minimum site-size of the
protein-nucleic acid complex, n = 15, and the size of
the binding site engaged in protein ssDNA interactions, m = 8. The helicase binds the ssDNA in a single orientation
with respect to the polarity of the sugar-phosphate backbone of the
ssDNA. The ssDNA-binding site, which encompasses 8 nucleotide residues,
is located on one side of the enzyme molecule, with the rest of the
protein matrix protruding over the extra 7 nucleotide residues, without
engaging in thermodynamically significant interactions with the DNA
(black ribbon) (a). When bound at the ends of the nucleic
acid, or in its center, the protein can occlude 8 or 15 nucleotides
(b). Therefore, this model would allow the binding of two
molecules of the PriA helicase to the ssDNA 24-mer and three molecules
of the enzyme to the 40-mer (c). However, as shown is this
work, this is not experimentally observed.
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Fig. 7.
Schematic model for the binding of the PriA
helicase to the ssDNA, based on the total site-size of the
protein-nucleic acid complex, n = 20, and the size of
the ssDNA-binding site engaged in protein-ssDNA interactions,
m = 8. The helicase binds the ssDNA in a single
orientation with respect to the polarity of the sugar-phosphate
backbone of the ssDNA. The ssDNA-binding site of the PriA helicase,
which encompasses only 8 nucleotide residues, is located in the center
of the enzyme molecule (a). The protein matrix protrudes
over 6 nucleotide residues on both sides of the ssDNA-binding site
without engaging in interactions with the nucleic acid (black ribbon).
When bound at the 5' or the 3' end of the nucleic acid, the protein
always occludes 14 nucleotide residues, whereas in the complex in the
center of the ssDNA oligomer, 20 nucleotide residues are occluded
(b). This model would allow the binding of only one molecule
of the PriA helicase to the 24-mer and only two molecules of the enzyme
to the 30-, 35-, and 40-mers (c). This is in complete
agreement with all of the experimental results described in this
work.
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Intrinsic Affinities of PriA-ssDNA Interactions--
Binding of a
single PriA molecule to 8-, 10-, 14-, 16-, 18-, 20-, 24-, and 26-mers
can be analyzed using a single-site-binding isotherm described by
Equation 5,
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(Eq. 5)
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where KN is the apparent binding constant
characterizing the affinity for a given ssDNA oligomer, containing
N nucleotide residues, and Fmax is
the maximum relative fluorescence increase. Notice the value of
KN contains a statistical factor resulting from the
fact that the number of nucleotide residues engaged in interactions
with the protein ssDNA-binding site, within the total site-size of the
PriA·ssDNA complex, m = 8. Knowing the number of
residues engaged in direct interactions with the ssDNA-binding site of
the enzyme allows us to properly extract the intrinsic binding
constant, Ki, for the ssDNA oligomers, which bind
only a single PriA molecule. By using the intrinsic binding constant,
Ki, the equation describing the binding of PriA to
ssDNA oligomers, which can accommodate only a single enzyme molecule,
is defined as shown in Equation 6.
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(Eq. 6)
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where N is the number of nucleotide residues in the
ssDNA oligomer. It should be pointed out that Equation 6 is different from the isotherm, typically applied in the binding of a large ligand
to a finite lattice, which takes into account the overlap of potential
binding sites, although the mathematical form is the same (38-40). The
difference results from the fact that instead of the total site-size of
the protein·ssDNA complex (n = 20), the number of the
nucleotide residues engaged in direct interactions with the
ssDNA-binding site of the protein, m, enters the equation (in the case of PriA, m = 8).
The solid lines in Fig. 1a are nonlinear least
square fits of the experimental binding isotherm for the PriA-24-mer
system using Equation 6, with N = 24 and
m = 8, which provide Ki = (4.4 ± 0.6) × 104
M1. The lines are parametric
plots where F, defined by Equation 6, is plotted
versus total protein concentration, PTot = PF + (i)NTot (Equation 4).
The values of the intrinsic binding constants, together with the
KN values for all studied ssDNA oligomers, are
included in Table I. The value of KN, within
experimental error, increases as the length of the ssDNA oligomers
increase. This results from the increasing value of the hidden
statistical factor in Equation 5. However, with the exception of the
shortest oligomer (8-mer), the intrinsic binding constant is centered
around the value of (5.5 ± 2) × 104
M1. Such behavior is expected for
the binding constant characterizing intrinsic interactions that should
be independent of the length of the nucleic acid oligomer, as long as
the oligomer is longer than m. Also, these data indicate
that there are no significant end effects in the PriA helicase binding
to the ssDNA.
Statistical Thermodynamic Models of the PriA Helicase Binding to
the ssDNA 30-, 35-, and 40-mers, Intrinsic Affinities, and
Cooperativities--
Quantitative analysis of the PriA helicase
binding to a 30-mer and longer oligomers is much more complex. The
partition function, ZN, for these PriA-ssDNA
oligomer systems must account for the potential overlap of the binding
sites and the possible cooperative interactions between the bound
protein molecules. The situation is additionally complicated by the
fact that the PriA protein can efficiently associate with ssDNA
oligomers, which are significantly shorter than the total site-size of
the protein·ssDNA complex. Thus, two enzyme molecules associate with
the 30-mer, although the length of the oligomer is shorter than the sum
of the two total site-sizes of the PriA·ssDNA complex (see above). Moreover, we know that the number of nucleotide residues engaged in
interactions with the ssDNA-binding site of the enzyme is
m = 8 and that the protein protrudes over a distance of
6 ± 1 nucleotides on both sides of the binding site (Fig. 7). For
instance, the PriA protein can bind to the 5' or 3' end of the ssDNA,
occluding only 14 nucleotide residues instead of 20 (Fig. 7).
The considered complex binding mechanism is unique and, to our
knowledge, never before analyzed. Such a binding system cannot be
directly treated by the exact combinatorial theory for large ligand
binding to a finite linear, homogeneous lattice that exclusively uses
the total site-size of the protein-lattice complex (38-40). The
partition functions for the 30-, 35-, and 40-mers,
ZN, have been obtained by examining all possible
states of the given PriA·ssDNA oligomer complexes. In the case of the
30-mer, the complete partition function of the PriA-30-mer system,
Z30, which takes into account the overlap of
potential binding sites and the formation of the PriA·ssDNA complexes
lower than the total site-size, as depicted in Fig. 7, is described by
Equation 7,
|
(Eq. 7)
|
where is the parameter characterizing the cooperative
interactions between bound protein molecules. Analogous partition functions for the 35- and 40-mers are shown in Equation 8,
|
(Eq. 8)
|
and Equation 9,
|
(Eq. 9)
|
The average degree of binding, i, is then obtained
by using the standard statistical thermodynamic expression, i = 
lnZN/lnPF (38-41).
For the binding of PriA to the 30-, 35-, and 40-mers, one obtains
Equations 10-12.
|
(Eq. 10)
|
|
(Eq. 11)
|
|
(Eq. 12)
|
The observed fluorescence increase, F, of
dA(pA)29, dA(pA)34, and
dA(pA)39 as a function of the PriA concentration is
then defined by Equations 13-15,
|
(Eq. 13)
|
|
(Eq. 14)
|
|
(Eq. 15)
|
where F1 and
Fmax are relative, molar fluorescence
increases accompanying the binding of the one and two PriA molecules to
the ssDNA oligomer, respectively. The values of
F1 and Fmax can be
independently determined from the dependence of the nucleic acid
fluorescence upon the average number of PriA molecules bound per
oligomer, i as depicted in Figs. 2b and
3b for the 30- and 40-mers. The plots are linear, indicating
that the same fluorescence increase accompanies the binding of the
first and the second PriA molecule to the 30- and 40-mers. The same is
true for the 35-mer (data not shown). As we pointed out, such dependence of the observed spectroscopic signal, as a function of the
degree of binding, should be determined and not a priori assumed (19, 29-32, 34, 35). For the 30-mer,
F1 = 1.25 and Fmax = 2.5. The solid lines in Fig. 2a are nonlinear
least square fits using Equations 7 and 13, with only two fitted
parameters Ki and . The obtained values are
Ki = (1.3 ± 0.3) × 105
M1 and = 11 ± 3.
Analogous analyses of the binding of the PriA protein to 35- and
40-mers (e.g. Fig. 3a) provide similar values of
the intrinsic binding constants, although a slightly lower
cooperativity parameter (Table II). A
similar value of Ki, obtained with ssDNA oligomers
of different lengths, indicates similar types of intrinsic interactions
between the PriA protein and the nucleic acid in the studied complexes.
This is predicted by the proposed model of the protein·ssDNA complex,
which takes into account the total site-size of the protein-nucleic
acid complex, n, the number of the nucleotide residues
engaged in interactions with the ssDNA-binding site, m, and
the location of the ssDNA-binding site within the protein matrix (Fig.
7, Equations 7-15). The obtained value of ranging between 11 ± 3 and 0.8 ± 0.3 also indicates that the binding of the PriA
helicase to the ssDNA is not accompanied by significant cooperative
interactions (see "Discussion").
View this table:
[in this window]
[in a new window]
|
Table II
Thermodynamic and spectroscopic parameters characterizing binding of
the PriA helicase to ssDNA oligomers that can accommodate two enzyme
molecules in buffer C (pH 7.0, 10 °C) containing 100 mM
NaCl
|
|
| |
DISCUSSION |
The physiological role of the PriA helicase is related to the
ability of the enzyme to interact with both ss- and dsDNA (1-3). Yet,
little is known about the quantitative molecular mechanism of the PriA
helicase-nucleic acid interactions. In this communication, we provide
extensive, quantitative studies of the stoichiometry, free energy of
binding, and cooperativity of the PriA protein interactions with the
ssDNA.
Application of Etheno Derivatives of the ssDNA to Quantitatively
Study Helicase-Nucleic Acid Complexes--
Quantitative studies of the
interactions of the PriA helicase with ssDNA are greatly facilitated by
the finding that binding of the protein to the fluorescent etheno
analog (A) of adenosine homopolymers is accompanied by a strong
nucleic acid fluorescence increase (19, 28-31). A similar effect of a
helicase on the fluorescence of the etheno derivatives has been
previously found in the case of the E. coli hexameric DnaB
helicase and was extensively used by us to quantitatively examine the
thermodynamics and kinetics of the DnaB helicase interactions with the
ssDNA (19, 29-32, 42).
The fluorescence of A is not very sensitive to the polarity of the
environment (25-28, 43). However, as compared with the free AMP,
the emission of etheno oligomers is predominantly quenched (8-10-fold), via the dynamic process of an intramolecular collision (36, 37, 44). The observed strong fluorescence increase of the etheno
derivative oligomers, upon binding to the PriA helicase, most probably
results from the large conformational change of the nucleic acid
leading to the increased separation and restricted mobility of the
nucleic acid bases in the complex with the enzyme (19, 20, 37). A large
fluorescence increase of A derivatives, in the complexes with both
PriA and DnaB helicases, indicates that the structural changes of the
ssDNA, induced by the complex formation with different helicases, are
similar. This is particularly interesting in light of the fact that
PriA and DnaB are fundamentally different in their helicase activity.
The DnaB protein is a 5' 3' helicase, i.e. it
unwinds the duplex DNA in a 5' direction, while PriA is a 3' 5'
helicase (1-3). Thus, independently of the direction of dsDNA
unwinding, a helicase induces very similar conformational changes in
the bound ssDNA.
The Single ssDNA-Binding Site of the PriA Helicase Encompasses
8 ± 1 Nucleotide Residues--
In the complex with the polymer
ssDNA, a large protein ligand will block the access of other protein
molecules to n number of nucleotide residues (38-40). This
nucleic acid fragment, a fundamental quantity for proper analysis of
the energetics and structure of the protein-nucleic acid complexes, is
referred to as the total site-size of the protein-nucleic acid complex
(31, 38-40). Because PriA-polymer ssDNA complexes precipitate at high
protein and nucleic acid concentrations, we could not quantitatively
determine the total site-size of the protein·ssDNA complex by
directly using the polymer nucleic acid. Nevertheless, the obtained
data with polymer ssDNA, limited to the protein concentrations where
the precipitation was not yet present (~4 × 105 M), already indicated that
the binding density (number of protein molecules bound per nucleotide
residue) reached the value of ~0.04. This value shows that the total
site-size of the PriA·ssDNA complex cannot be larger than ~25
nucleotides (38-40).
The major aspect of the experimental strategy used in this work is the
application of the large series of ssDNA oligomers of well defined
length. Oligomers range from a length shorter than the number of
nucleotide residues encompassed by the ssDNA-binding site of the enzyme
to ssDNA oligomers that can accommodate two PriA molecules. Such an
approach dramatically increases the resolution of the binding
experiments and allows us to determine not only the total site-size of
the protein·ssDNA complex, n, but also the number of
nucleotide residues directly engaged in interactions with the
ssDNA-binding site of the protein, m. In other words, it
allows us to determine the size of the ssDNA-binding site of the
enzyme. Only when these two parameters are known can the proper statistical factors be obtained and the intrinsic binding constants extracted (Tables I and II). Moreover, the possibility of analyzing the
stoichiometries of protein·ssDNA complexes with different oligomers
enables us to get insight into the location of the ssDNA-binding site
within the protein matrix.
As we discussed above, in direct interactions, the ssDNA-binding site
of a protein may engage a significantly smaller number of nucleotide
residues than the total site-size of the complex. This number of
nucleotide residues encompassed by the ssDNA-binding site of the
protein, m, can be estimated in experiments with short ssDNA
oligomers. The minimum value of m corresponds to the length of the shortest ssDNA oligomer that still forms a complex with the
protein, with an affinity similar to the affinity of longer oligomers.
In other words, such an oligomer, although shorter than the total
site-size, must form all crucial, interacting contacts with the
ssDNA-binding site of the enzyme (31). By using this approach, we
established that the ssDNA-binding site of the E. coli
replicative helicase, the DnaB protein, encompasses only 6-7
nucleotide residues although the total site-size of the protein·ssDNA complex is 20 ± 3 nucleotides (31). In the case of the PriA helicase, in order to efficiently form a complex with the ssDNA-binding site, the ssDNA oligomer must have a length of m = 8 ± 1 nucleotide residues, a value significantly shorter than the
total site-size. Thus, the difference of 2 nucleotide residues between
6- and 8-mers makes the affinity of the shorter oligomer for the
binding site barely detectable (Fig. 5). However, the same difference
between 8-and 10-mers does not lead to any decrease of the free energy of binding (Table I).
For the examined series of ssDNA oligomers, the PriA helicase forms
complexes with a stoichiometry of 1:1 with oligomers of 8-26
nucleotide residues in length (Fig. 4). As discussed above, the number
of nucleotide residues encompassed by the ssDNA-binding site of the
enzyme, m, is the minimum number of nucleotides necessary for the protein and a nucleic acid to efficiently form a complex. Knowing the value of m allows us to properly address the
statistical factor in these complexes and to extract the intrinsic
binding constant Ki that should be independent of
the length of the nucleic acid. Thus, the values of the apparent
binding constants for studied oligomers, obtained using Equation 5,
show, within experimental accuracy, an increasing trend with the
increasing length of the studied oligomer, as a result of the
increasing value of the statistical factor (Table I). However,
intrinsic binding constants, determined using Equation 6, which take
into account the statistical factor, show no such trend as a function of the length of the ssDNA and are centered around (5.5 ± 2) × 104 M1. This is
exactly what is expected for the binding constant that characterizes
intrinsic interactions between the protein and the nucleic acid, not
affected by the statistical factors.
The Total Site-size of the PriA Helicase Complex with ssDNA Is
20 ± 3 Nucleotide Residues--
A transition from the
stoichiometry of 1:1 to the stoichiometry of two PriA molecules bound
per ssDNA occurs between the complexes with 26- and 30-mers (Fig. 4).
The fact that two PriA molecules bind to the 30-mer would suggest that
the total site-size of the enzyme·ssDNA complex is 15 nucleotides,
yet only two protein molecules bind to the 35- and 40-mers, which would
suggest a site-size larger than 15. However, knowing that the
ssDNA-binding site on the protein binds only 8 nucleotide residues
opens the possibility of more accurately determining the total
site-size of the protein·ssDNA complex. Moreover, the obtained
extensive data on the stoichiometries of the enzyme-ssDNA oligomer,
over the entire spectrum of the length of the oligomer, provide insight
into the structure of the protein-nucleic acid complex.
It is clear that if the ssDNA-binding site of the PriA helicase is
located at one end of the protein molecule and the site-size is
n = 15, then two molecules of the enzyme would bind to
the 24- and 26-mers (Fig. 6, a-c). This is because the
first bound PriA can form a complex with the nucleic acid where only 15 residues are blocked, with 9 or 11 residues remaining free. These ssDNA fragments would be long enough to accommodate another protein molecule
that can bind, with a very similar intrinsic binding constant, to a
stretch of only 8 residues (Fig. 6c). The same analysis
shows that if the total site-size is 15 residues long and the
ssDNA-binding site is located as depicted in Fig. 6, then the 40-mer
should bind three PriA molecules. However, the experimental data show
that only one and two PriA molecules bind to the 24- and 40-mers, thus
contradicting this model.
The schematic model of the PriA protein·ssDNA complex, which agrees
with all thermodynamic data described in this work, is shown in Fig. 7.
The ssDNA-binding site, encompassing 8 nucleotide residues, is located
in the center of the protein molecule. On both sides of the
ssDNA-binding site the protein matrix protrudes over a distance of 6 residues, giving a total site-size of the protein·ssDNA complex as
n = 20. A characteristic feature of this model is that
the bound protein always blocks either 20 or a stretch of 14 nucleotide
residues in the complex with the ssDNA (Fig. 7b). As a
result, in the complex with the 24- and 26-mers, the bound PriA blocks
a stretch of at least 14 residues, leaving only 11 and 12 residues
free, respectively. This is not enough for the second PriA molecule to
associate with the ssDNA. With the 30-mer, the protein can form a
complex where the stretch of 16 residues is free, making the binding of
the second protein molecule possible (Fig. 7c). In the case
of the 40-mer, two PriA molecules can form a complex with the oligomer
where 34 residues are blocked, leaving only a stretch of 6 residues
free. Once again, this is not enough for the third molecule of the
enzyme to associate with the nucleic acid (Fig. 4).
As we mentioned above, independence of intrinsic binding constants
characterizing interactions of the enzyme with the ssDNA oligomers that
accommodate only one protein molecule upon the oligomer length strongly
support the proposed model of the PriA·ssDNA complex (Table I).
Additional support of the model of the PriA·ssDNA complex, shown in
Fig. 7, comes from the determined intrinsic binding constants for
oligomers that bind two PriA molecules, 30-, 35-, and 40-mers. The
determined values of Ki are similar for all these
oligomers. Moreover, the values of Ki are also
similar to the values of Ki determined for the short
oligomers (Tables I and II). This could only happen if the statistical
factors that enter the partition function and that are strongly
dependent on the total site-size and size of the ssDNA-binding site are
correctly determined (Equations 7-15).
There Are Very Weak Cooperative Interactions in the Binding of the
PriA Helicase to the ssDNA--
Binding of the PriA helicase to the
ssDNA is characterized by the cooperativity parameter, ~0.8-11
which indicates very weak cooperative interactions between bound
protein molecules (Table II). Similar low values of characterize
the binding to the ssDNA of another E. coli replicative
helicase, the DnaB protein (19, 34). The DnaB protein is the only other
helicase for which the value of is known. We believe that the fact
that both the DnaB hexamer and the PriA monomer are unable to form long protein clusters, when bound to the ssDNA, may reflect a general aspect
of functioning of the replicative helicase (19, 34). The unwinding of
the dsDNA at the junction between ss- and dsDNA does not require
clusters of helicase molecules. Very weak or nonexistent
protein-protein interactions may be a simple evolutionary adaptation of
a replicative helicase, which allows the enzyme to perform functions
requiring only a single protein molecule (19, 20).
| |
ACKNOWLEDGEMENT |
We thank Gloria Drennan Davis for help
in preparing the manuscript.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM-46679 and GM-58675 (to W. B.).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.
To whom correspondence should be addressed: Dept. of Human
Biological Chemistry and Genetics, the University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX
77555-1053.
Published, JBC Papers in Press, June 29, 2000, DOI 10.1074/jbc.M004104200
| |
ABBREVIATIONS |
The abbreviations used are:
PAS, primosome
assembly site;
A, 1,N6-etheno adenosine;
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA.
| |
REFERENCES |
| 1.
|
Kornberg, A.,
and Baker, T. A.
(1992)
DNA Replication
, pp. 275-306, W. H. Freeman & Co., San Francisco, CA
|
| 2.
|
Marians, K. J.
(1992)
Annu. Rev. Biochem.
61,
673-719
|
| 3.
|
Marians, K. J.
(1999)
Prog. Nucleic Acids Res. Mol. Biol.
63,
39-67 |
TOP
ABSTRACT
INTRODUCTION
