 |
INTRODUCTION |
Polymerase
is one of a number of recognized DNA-directed
polymerases of the eukaryotic nucleus (1-5). The enzyme has a very
specialized function in mammalian cell repair machinery. The activities
of pol
1 have been
proposed to include the gap filling synthesis involved in mismatch
repair (4, 6-8), in the repair of monofunctional adducts, UV damaged
DNA, and abasic lesions in DNA (9-14). Thus, a processive "gap
fillings" synthesis, observed in vitro, by rat pol
on
gapped DNAs, is consistent with the proposed in vivo activities (2, 6-8). A characteristic feature of rat pol
is a
"simplified" repertoire of activities. The enzyme is lacking intrinsic accessory activities, such as 3'- or 5'-exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1-4, 6, 7). Such
limited activities reflect the very specialized function of the
polymerase in the DNA metabolism.
The crystal structure of the rat pol
enzyme revealed a typical
polymerase fold, a thumb, palm, and fingers, due to its resemblance to
the human hand (15-17). However, what distinguishes the pol
structure from other polymerases is the presence of a small 8-kDa
domain that is connected with the tip of the fingers through a tether
of 14 amino acids (15-17). Solution studies showed that the 8-kDa
domain has significant affinity for the single-stranded (ss) DNA,
indicating that the domain plays a key role in recognition of this
nucleic acid conformation, i.e. it is the template-binding domain (15, 18, 19, 21, 22). The active site of the DNA synthesis and
the dsDNA affinity were proposed to reside predominantly in the large
31-kDa catalytic domain (15, 18, 19, 23).
Recently, we examined interactions of rat and human polymerase
with
the ssDNA (20, 24). Our data showed that rat pol
binds the ssDNA in
two binding modes which differ in the number of occluded nucleotide
residues, the (pol
)16 and (pol
)5
binding mode. The binding modes differ in affinities and abilities to induce conformational changes in the ssDNA (20). Thus, the intrinsic affinity of the enzyme in the (pol
)16 binding mode is
approximately an order of magnitude higher than the affinity in the
(pol
)5 binding mode. On the other hand, when bound in
the (pol
)5 binding mode, the enzyme induces much more
profound structural changes in the ssDNA, suggesting strong base-base
separation and DNA immobilization in the complex (20). The obtained
results also indicate that in the (pol
)16 binding mode,
both the 8- and 31-kDa domain of the enzyme, are involved in
interactions with the ssDNA. In the (pol
)5 binding
mode, the 8-kDa domain predominately is engaged in interactions with
the nucleic acid (20).
Elucidation of the pol
-ssDNA recognition processes constitutes a
first step toward understanding the molecular mechanism of the
polymerase. However, in vivo, the enzyme also recognizes the
more complex DNA substrates, gapped DNAs with different ssDNA gaps, and
template-primer substrates resulting from the DNA damage (4, 21-23).
Such interactions are certainly more complex and involve the
simultaneous interactions of the polymerase with both the ss- and
dsDNAs (4, 21, 22). Moreover, steady-state kinetic analysis suggests
that the presence of the 5'-terminal phosphate group, downstream from
the primer, amplified the enzyme affinity for the gapped DNA (22, 23).
Despite the paramount importance for understanding the polymerase
mechanism, the direct analysis of the energetics of rat pol
interactions with the template-primer and the gapped DNA substrates has
not been quantitatively addressed before.
In this article, we report the analysis of rat pol
interactions with different DNA substrates, including the
primer-template, with different ssDNA extensions and gapped DNA
substrates, with different gap sizes, using the quantitative
fluorescence titration technique (20). We provide direct evidence that
rat pol
binds the template-primer DNA and the gapped DNA substrates
with stoichiometries much higher than predicted by the crystallographic
structure of the co-complexes with the DNA. These high stoichiometries
can be accounted for by the fact that, in solution, the enzyme uses two
binding modes in its interactions with the ssDNA (20). The data
indicate that the 8-kDa domain of the enzyme is engaged in interactions
with the dsDNA, downstream from the primer. The 5'-terminal phosphate,
downstream from the primer, has little effect on the enzyme affinity
although it affects the structure of the ssDNA in the complex. The
affinity and stoichiometry of rat pol
complexes with gapped DNAs
are not affected by the decreasing size of the ssDNA gap. A plausible
model of the ssDNA gap recognition by rat pol
is proposed.
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EXPERIMENTAL PROCEDURES |
Reagents and Buffers--
All chemicals were reagent grade. All
solutions were made with distilled and deionized >18M
(Milli-Q
Plus) water. Buffer C is 10 mM sodium cacodylate adjusted
to pH 7.0 with HCl, 1 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol. The
temperatures and concentrations of NaCl in the buffer are indicated
throughout the text.
Rat Polymerase--
Rat pol
was purified using a previously
published procedure (14, 17, 18, 20). The concentration of the protein
was determined using the extinction coefficient
280 = 2.1 × 104 cm
1
M
1, determined by the approach based on the
Edelhoch method (20, 24-27).
Nucleic Acids--
All nucleic acids were purchased from Midland
Certified Reagents (Midland, TX). The etheno-derivatives of the nucleic
acids were obtained by direct synthesis using
A phosphoramidite (28, 29). Concentrations of all ssDNA oligomers have been
spectrophotometrically determined, using the nearest neighbor analysis
(30-32). The template-primer and gapped DNA substrates were
obtained by mixing the proper oligomers at given concentrations,
warming the mixture for 5 min at 95 °C, and slowly cooling for a
period of ~3-4 h (28, 32, 33).
Fluorescence Measurements--
All steady-state fluorescence
titrations were performed as previously described by us (20, 24,
32-35). The binding was followed by monitoring the fluorescence of the
ethenoadenosine residues placed in the ssDNA extensions of the
template-primer or gapped DNA substrates. Computer analyses of the
binding isotherms were performed using Mathematica (Wolfram, IL) or
KaleidaGraph (Synergy Software, Reading, PA). The relative
fluorescence increase of the nucleic acid,
F, upon
binding rat pol
is defined as
F = (Fi
Fo)/Fo,
where Fi is the fluorescence of the sample at a
given titration point "i," and Fo is
the initial value of the fluorescence of the sample (32-35).
Quantitative Determination of Stoichiometries and Binding
Isotherms of Rat Pol
-DNA Complexes--
In this work, we followed
the binding of rat pol
to the various DNA substrates by monitoring
the fluorescence increase,
F, of their etheno-derivatives
upon the complex formation. The method to obtain quantitative estimates
of the average degree of binding,
vi (number of
protein molecules bound per DNA substrate) and the free protein
concentration, PF, has been previously described in
detail by us (20, 24, 28, 29, 32-35). Briefly, the experimentally
observed F has a contribution from each of the different
possible i complexes of rat pol
with a ssDNA.
Thus, the observed fluorescence increase is functionally related to
vi by (32-35)
|
(Eq. 1)
|
where
Fimax is the
molecular parameter characterizing the maximum fluorescence increase of
the nucleic acid with rat pol
bound in complex i. The same value of
F 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,
vi and the free pol
concentration,
PF, must be the same. The values of
vi and PF are then related to
the total protein concentrations,
PT1 and
PT2, and the total
nucleic acid concentrations,
NT1 and
NT2, at the same value of
F, by
|
(Eq. 2)
|
|
(Eq. 3)
|
where x = 1 or 2 (32-35).
 |
RESULTS |
Binding of Rat pol
to Template-Primer DNA Substrates with
5-Nucleotide Long ssDNA Extension--
The two binding modes, (pol
)16 and (pol
)5, which rat pol
forms
with the ssDNA, differ by the number of the occluded nucleotide
residues in the protein-nucleic acid complex (20). To determine the
effect of the structure of a DNA substrate on the energetics of the
enzyme binding in different binding modes, interactions of the enzyme
with the template-primer DNA have been examined using DNA substrates,
in which the ssDNA extension can accommodate the enzyme in the (pol
)16 and/or the (pol
)5 binding mode.
Template-primer DNA substrates, used in these studies, are depicted in
Fig. 1. The duplex part of each substrate
is 10 bp long and is located at the 5' or 3' end of the template
strand. The length of the dsDNA part has been selected on the basis of the co-crystal structure of rat pol
with the template-primer DNA
indicating that the polymerase encompasses up to ~7 base pairs (bp)
of the dsDNA (16, 17). The ssDNA extensions are 5 or 15 bases long and
correspond to the site sizes of 5 ± 2 and 16 ± 2 nucleotide
residues in the (pol
)5 or (pol
)16
binding mode (20). A stretch of fluorescent ethenoadenosine residues
(
A) are placed in each of the ssDNA extensions, directly from the dsDNA part which provides the fluorescence signal to monitor the complex formation (20, 24, 32-35).

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Fig. 1.
Template-primer DNA substrates used to
examine the energetics of rat pol binding to template-primer
DNAs. The duplex part of each substrate is 10 bp long and is
located at the 5' or 3' end of the template strand. The ssDNA
extensions have 5 (substrates A and B) or 15 (substrates C, D, and E)
nucleotide residues which correspond to the site-size of 5 ± 2 and 16 ± 2 nucleotide residues, occluded by the enzyme in the
complex with the ssDNA complex in the (pol )5 or (pol
)16 binding mode (20). The ssDNA extensions of the
template strand have a stretch of five fluorescent ethenoadenosine
residues ( A) adjacent directly to the dsDNA. Substrate B has the 3'
ssDNA extension with four As. The presence of As provides the
fluorescence signal to monitor the complex formation with the
polymerase (30-35). Substrate E is the same as D, but contains a 5'
terminal phosphate group on the oligonucleotide in the dsDNA part at
the 5' end of the template strand.
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First, we examine interactions of rat pol
with template-primer DNAs
which potentially can accommodate the polymerase only in the (pol
)5 binding mode on their ssDNA extensions,
i.e. the extensions are 5 nucleotide residues long (Fig. 1;
substrates A and B). Substrate A has a ssDNA extension at the 5' end of
the template, i.e. it resembles a natural template-primer
DNA (16, 17). Substrate B has a ssDNA extension at the 3' end of the template. Fluorescence titrations of the template-primer substrate, containing the ssDNA extension with 5 nucleotide residues at the 5' end
of the template, with rat pol
, at two different nucleic acid
concentrations, in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, are shown in Fig.
2a. The relative nucleic acid
fluorescence increase reaches the value of 1.1 ± 0.1. At a higher
DNA substrate concentration, a given fluorescence increase,
F, is reached at higher polymerase concentrations, due to
the enzyme binding to an extra DNA in the solution.

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Fig. 2.
a, fluorescence titrations
( ex = 325 nm, em = 410 nm) of the
template-primer DNA substrate (Fig. 1; substrate A), containing the
ssDNA extension with five nucleotide residues at the 5' end of the
template, with rat pol , in buffer C (pH 7.0, 10 °C), containing
100 mM NaCl, at two different concentrations of the nucleic
acid: ( ) 2.22 × 10 7 M; ( )
4.44 × 10 7 M. The solid
lines are nonlinear least square fits of the fluorescence
titration curves, according to the model of the two binding sites,
defined by Equation 6. The lines are plotted using a single
set of parameters: KDS = 1.8 × 106 M 1, K5 = 2.6 × 106 M 1,
FDS = 0.7, and F5 = 0.4 (details in text). Notice that for this substrate,
K5 characterizes binding to the ss-ds DNA
junction and is referred to in the text as KJ.
b, the dependence of the relative fluorescence increase,
F, upon the degree of binding, vi,
of the rat pol -DNA substrate complex. The values of
vi have been determined using the quantitative
method described under "Experimental Procedures." The solid
lines are the limiting slopes of the two binding phases. The
dashed line is an extrapolation of the degree of binding to
the maximum value of the observed fluorescence increase
Fmax = 1.1 ± 0.1 which provides the
maximum stoichiometry of 2 ± 0.3 of the rat pol -DNA substrate
complex.
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|
To quantitatively obtain the average degree of binding,
vi, independent of any assumption about the
relationship between the observed signal and the degree of binding, the
titration curves in Fig. 2a have been analyzed, using the
approach outlined under "Experimental Procedures" (32-35). Fig.
2b shows the dependence of the observed relative
fluorescence increase as a function of the average degree of binding,
vi. The plot is nonlinear and shows two binding
phases. In the first high-affinity phase, characterized by the relative
fluorescence increase 0.4 ± 0.1, the degree of binding reaches
the value of 1 ± 0.2 which shows that in the first step a single
molecule of the polymerase associates with the template-primer
substrate. In the second phase, in the high protein concentration
region, extrapolation of
vi to the maximum
fluorescence increase,
Fmax = 1.1 ± 0.1, provides a value of
vi = 2 ± 0.2.
Analogous fluorescence titrations have been performed with the
template-primer substrate, containing the ssDNA extension at the 3' end
of the template (Fig. 1; substrate B) (data not shown). Similar to
substrate A, the maximum relative increase of the nucleic acid
fluorescence reaches the value of 1.2 ± 0.1 (Table
I). In the first binding phase,
characterized by a relative fluorescence increase of 0.6 ± 0.1, the degree of binding reaches the value of 1 ± 0.2, thus, showing
that in the first step a single molecule of the polymerase associates
with this template-primer substrate. In the low-affinity phase,
extrapolation of
vi to the maximum fluorescence
increase,
Fmax = 1.2 ± 0.1, provides a
value of
vi = 2.3 ± 0.2.
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Table I
Thermodynamic and spectroscopic parameters for rat pol binding to
the template-primer DNA substrates, which have ssDNA extensions with 5 nucleotide residues (Fig. 1; substrates A and B) in buffer C (pH 7.0, 10 °C) containing 100 mM NaCl
The errors are standard deviations determined using three to four
independent titration experiments.
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The obtained stoichiometries are surprisingly high. Recall, with a
template-primer DNA similar to substrate A (Fig. 1), only one rat pol
molecule has been found bound to the DNA in the crystal (16).
However, the stoichiometries obtained for both substrates can be
understood if one takes into account that the ssDNA extensions are long
enough to accommodate only one polymerase molecule in the (pol
)5 binding mode, while another enzyme molecule can
associate with the dsDNA part of the DNA (16, 20). In other words, the
considered DNA substrates have two different binding sites. Because all
DNA substrates examined in this work (Figs. 1 and 5) have the same
dsDNA part and different ssDNA extensions, the binding to the dsDNA
parts should be characterized by the same affinity for all studied
substrates (see below).
The two binding sites on the considered DNA substrates differ
structurally. Therefore, the association of rat pol
with these sites can be analyzed as binding to two discrete binding sites (36).
The partition function, Z, and the average degree of
binding,
vi, are then described as,
|
(Eq. 4)
|
and
|
(Eq. 5)
|
where PF is the free rat pol
concentration,
KDS and K5 are the
binding constants for the dsDNA part and to the ssDNA extension in the
(pol
)5 binding mode, respectively. The observed
relative fluorescence change,
F, of the nucleic acid fluorescence is then defined as,
|
(Eq. 6)
|
where
FDS and
F5 are fluorescence increases accompanying
the binding of rat pol
to the dsDNA part and the ssDNA extension, respectively. The values of
FDS and
F5 can be determined from the plots of the
observed fluorescence change as a function of the average degree of
binding,
vi and the known maximum observed
fluorescence change
Fmax =
FDS + F5 (Fig. 2;
Table I).
The solid lines in Figs. 2a are computer
fits using Equations 4-6 which provide KDS = (1.8 ± 0.5) × 106 M
1 and
K5 = (4.6 ± 0.6) × 106
M
1, for the DNA substrate with the 5' ssDNA
extension, and KDS = (2 ± 0.5) × 106 M
1 and
K5 = (3 ± 0.6) × 105
M
1, for the nucleic acid with the 3' ssDNA
extension, respectively (Table I). Notice, because both DNA substrates
have the same dsDNA part, the values of KDS are
virtually the same, reflecting the presence of the same binding site on
both nucleic acids. The very similar values of
FDS indicate that the presence of the protein
bound to the dsDNA affects the ssDNA conformation to a similar extent
in both substrates.
However, the determined values of K5 differ
significantly between the two nucleic acids (Table I). Previous studies
indicated that rat pol
binds template-primer and gapped DNA
substrates in a defined orientation, with the small 8-kDa domain facing
the 5' end of the template strand and the large 31-kDa domain facing the 3' end of the template strand (4, 11). Because both substrates differ only in the location of the dsDNA, with respect to the ssDNA
extension, such a large difference between the values of K5 indicates that the enzyme binds differently
to the 5' than to the 3' extension.
In the case of the DNA substrates with the 3' extension, rat pol
can only form the (pol
)5 binding mode (20). However, K5 is by factor of ~ 15 higher than the same
parameter, K5 = (2±1)×104M
1 characterizing the
affinity of the enzyme for the ssDNAoligomer, d
A(p
A)15, indicating that the 8-kDa domain of the
enzyme is engaging in interactions with the dsDNA part of the substrate (see below) (20). In the case of the DNA substrate with the 5'extension, the value of K5 is ~2 orders of
magnitude higher than the in trinsic binding constant for the (pol
)5 binding mode determined for the ssDNA oligomer (20).
Also, the value of
F5 is only 0.35±0.1
(Table I) indicating much less pronounced structural changes in the
ssDNA, as expected for the complex where the small 8-kDa domain is
engaged in interactions with the ssDNA in the (pol
)5
binding mode (
F5=1±0.1) (20).
Thus, these very different values of interaction and
spectroscopic parameters strongly suggest that, where the ssDNA
extension is located at the 5' end of the template, the polymerase does not form the (pol
)5 binding mode, but rather binds the
ss-dsDNA junction of the substrate, using the large 31-kDa catalytic
domain, as in the crystal structure of the analogous co-complex (16,17) (see "Discussion"). Therefore, we will refer to the parameters characterizing this particular complex as KJ and
FJ, rather than K5 and
F5 (Table I). Notice that the value of
KJ is virtually the same as the value of
KDS, characterizing the binding of the enzyme to
the dsDNA where the 31-kDa domain is predominantly involved (19) (Table
I).
Binding of Rat Pol
to Template-Primer DNA Substrates with a
15-Nucleotide Residue Long ssDNA Extension: Statistical Thermodynamic
Model--
The interactions of rat pol
with template-primer DNAs
having ssDNA extensions which can accommodate the polymerase not only in the (pol
)5 but also in (pol
)16
binding mode have been examined with the DNA substrates C and D in Fig.
1. Substrate C has a ssDNA extension at the 5' end of the template.
Thus, the possible complexes with this substrate include formation of
the (pol
)16 binding mode or a complex in which the
8-kDa domain of the enzyme can interact with the ssDNA of the template,
while the 31-kDa catalytic domain is engaged in interactions with the
ss-ds DNA junction (16). We refer to the latter complex as the
template-primer complex. Substrate D has a ssDNA extension at the 3'
end of the template, which prevents the enzyme from simultaneously
engaging the 8-kDa domain in interactions with the ssDNA and the 31-kDa domain in interactions with the ss-ds DNA junction in any of the possible complexes.
Fluorescence titrations of the template-primer substrate, containing a
ssDNA extension at the 5' end of the template with rat pol
, at two
different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C),
containing 100 mM NaCl, are shown in Fig.
3a. The relative increase of
the nucleic acid fluorescence reaches the value of 4.4 ± 0.2. Fig. 3b shows the dependence of the observed relative
fluorescence increase,
F, as a function of the average degree of binding,
vi, of the enzyme. The plot is
nonlinear and shows two binding phases. In the high-affinity phase, the degree of binding reaches the value of 1 ± 0.2 showing that, in this step, a single molecule of the polymerase associates with the
template-primer DNA substrate. In the second phase, in the high protein
concentration region, extrapolation of
vi to the
maximum fluorescence increase,
Fmax = 4.4 ± 0.2, provides a value of
vi = 4.2 ± 0.3.

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Fig. 3.
a, fluorescence titrations
( ex = 325 nm, em = 410 nm) of the
template-primer DNA substrate (Fig. 1; substrate C), having a ssDNA
extension with 15 nucleotide residues at the 5' end of the template,
with rat pol in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, at two different concentrations of the nucleic
acid (substrate): ( ) 4.5 × 10 7 M;
( ) 8.9 × 10 7 M. The solid
lines are nonlinear least-square fits of the fluorescence
titration curves according to the statistical thermodynamic model
defined by Equations 7-11. The model takes into account the
overlap of potential binding sites and cooperative interactions in the
formation of the (pol )5 binding mode on the ssDNA
extension, binding of the enzyme to the dsDNA part of the DNA
substrate, and formation of the template-primer complex, in which both
the 8- and 31-kDa domain of the protein are engaged in interactions
with the ssDNA and the ss-ds DNA junction, respectively (details in
text). The lines are plotted using a single set of
parameters (Table II). b, the dependence of the relative
fluorescence increase, F, upon the degree of binding,
vi, of the rat pol -DNA substrate complex. The
values of vi have been determined using the
quantitative method described under "Experimental Procedures." The
solid lines are the limiting slopes of the two binding
phases. The dashed line is an extrapolation of the degree of
binding to the maximum value of the observed fluorescence increase,
Fmax = 4.4 ± 0.2, which provides the
maximum stoichiometry of 4.2 ± 0.3 of the rat pol -DNA
substrate complex. c, fluorescence titrations the
template-primer DNA substrate (Fig. 1; substrate D) having the ssDNA
extension with 15 nucleotide residues at the 3' end of the template,
with rat pol in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, at two different concentrations of the nucleic
acid (substrate): , 4.5 × 10 7 M;
, 1.5 × 10 6 M. The solid
lines are nonlinear least-square fits of the fluorescence
titration curves according to the statistical thermodynamic model
defined by Equations 7-11. The model takes into account the overlap of
potential binding sites and cooperative interactions in the formation
of the (pol )5 binding mode on the ssDNA extension,
formation of the (pol )16 binding mode, and binding of
the enzyme to the dsDNA part of the DNA (details in text) (Table II).
d, the dependence of the relative fluorescence increase,
F, upon the degree of binding, vi,
of the rat pol -DNA substrate (Fig. 1; substrate D) complex. The
solid lines are the limiting slopes of the two binding
phases. The dashed line is an extrapolation of
vi to the maximum value of the observed
fluorescence increase, Fmax = 4.1 ± 0.2, which provides the maximum stoichiometry of 4.3 ± 0.3.
|
|
As we mentioned above, the high affinity step may correspond to either
the exclusive binding of the enzyme to the ssDNA in the (pol
)16 binding mode or to the simultaneous binding of the pol
molecule to the ssDNA of the template and the ss-ds DNA junction of the template-primer substrate. However, the affinity of the
(pol
)16 binding mode formed exclusively with the ssDNA is ~2 orders of magnitude lower than the affinity of the observed first binding phase in Fig. 3b (20) (see below). Such a
large difference in affinities excludes the formation of only the (pol
)16 binding mode with the ssDNA extension (20). Thus,
the data indicate that the high affinity step must predominantly
correspond to the formation of the template-primer complex. Because the
length of the ssDNA is 15 nucleotide residues, the 8-kDa domain can
interact with the ssDNA of the template, while the large 31-kDa domain is engaged in interactions with the ss-ds DNA junction of the substrate
(16).
The results show that, at saturation, four rat pol
molecules
bind to the considered DNA substrate (Fig. 3b; Table
II). Once again, such high stoichiometry
is fully understandable in the context of different ssDNA enzyme
binding modes and the structure of the DNA substrate (20). Notice, the
ssDNA with a length of 15 nucleotide residues can accommodate, at
saturation, three pol
molecules in the (pol
)5
binding mode. As the enzyme concentration increases, the negative
lattice entropy factor becomes very large (20, 24, 37, 38).
At high pol
concentrations, the template-primer complex is replaced
by the (pol
)5 binding mode with three enzyme molecules
associated with the ssDNA extension. The existence of mixed complexes
is not possible because the formation of the template-primer complex
and the binding in the (pol
)5 binding mode on a stretch of ssDNA with 15 nucleotide residues are mutually exclusive (20). The
ssDNA extension of the substrate cannot accommodate a rat pol
molecule bound in the template-primer complex and another one in the
(pol
)5 binding mode. Association of the fourth rat pol
molecule corresponds to the binding of one enzyme molecule to the
dsDNA part of the template-primer substrate, as determined for the DNA
substrates with the ssDNA of only 5 nucleotide residues in length (Fig.
2; Table I).
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Table II
Thermodynamic and spectroscopic parameters for rat pol binding to
template-primer DNA substrates having ssDNA extensions with 15 nucleotides (Fig. 1; substrates C, D, and E in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl
Errors are standard deviations determined using three to four
independent titration experiments.
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The considered interacting system is very complex. In the
simplest approach, the formation of the template-primer complex and
binding to the dsDNA part of the substrate are independent, and can be
described by a partial partition function, Z1
as,
|
(Eq. 7)
|
where KTP and KDS
are binding constants characterizing the formation of the
template-primer complex and binding to the dsDNA part,
respectively. As the protein concentration increases, the template-primer complex is replaced by the (pol
)5
binding mode while binding to the dsDNA remains unaffected. The part of
the total partition function, which describes the binding of
rat pol
in the (pol
)5 binding mode can be
formulated using the Epstein combinatorial theory for large
ligand binding to a finite, one-dimensional lattice (20, 24,
39). Thus, the complete partition function of the rat pol
-template-primer system, ZTP, is
then as shown,
|
(Eq. 8)
|
where K5 is the intrinsic binding constant
of rat pol
in the (pol
)5 binding mode,
is the
parameter characterizing cooperative interactions between the bound pol
molecules in this binding mode, g is the maximum number
of rat pol
molecules which can bind to the ssDNA extension, at
saturation (in our case g = 3), k is the
number of protein molecules bound at a given PF, and
j is the number of cooperative contacts between the
k bound rat pol
molecules in a particular configuration
on the lattice (20, 24, 39). The factor SM (k, j)
is the number of distinct ways that the k ligands bind to a
lattice, with j cooperative contacts, and is defined by (39)
in Equation 9.
|
(Eq. 9)
|
The total degree of binding,
vi, is then
defined as Equation 10.
|
(Eq. 10)
|
The observed fluorescence increase,
F, is described
by,
|
(Eq. 11)
|
where
FDS is the molar fluorescence
increase accompanying the binding of the polymerase to the dsDNA part
of the substrate;
FTP and
F5 are molar fluorescence increases
accompanying the formation of the template-primer complex and the (pol
)5 binding mode, respectively. It should be pointed out
that the quantity,
F5, is averaged over all
33 possible complexes of the enzyme with the ssDNA extension in the
(pol
)5 binding mode. Because the number of formed
possible complexes is very large, such averaging does not introduce a
significant error in the determination of the interaction parameters
(20, 24).
There are four interaction parameters, KTP,
KDS, K5, and
and
three fluorescence changes accompanying the formations of different complexes
FTP,
FDS,
and
F5, which describe the binding process. Because, the substrate has the same dsDNA part as in the previously examined DNA substrates with the ssDNA extension of 5 nucleotide residues in length (Fig. 1), we assign KDS = (2 ± 0.5) × 106 M
1
and
FDS = 0.6 ± 0.05 to this binding
process. The remaining five parameters are still a formidable number of
parameters, which precludes any attempt to obtain these quantities in a
single fitting procedure.
The determination of all interaction and spectroscopic parameters of
this very complex binding system can be achieved by applying the
following strategy. Inspection of the isotherms in Fig. 3, a
and b, shows that, due to its much higher macroscopic
affinity, the formation of the template-primer complex is significantly separated from the binding to the dsDNA and the (pol
)5
mode on the protein concentration scale. This separation of the binding processes allows us to independently estimate
FTP as the slope,
FTP = 
F/
(
vi),
of the initial part of the plot in Fig. 3b which provides
FTP = 1.9 ± 0.1. Also, because
initially the association of the enzyme with the DNA is completely
dominated by the formation of the template-primer complex, we can
determine the value of KTP from the initial
dependence of
vi, as a function of the protein
concentration (plot not shown). The obtained estimate is
KTP = (4 ± 2) × 107
M
1. The estimate of the average value of
F5 is based on the fact that the final
complex, at saturation, must contain one rat pol
bound to the dsDNA
and three pol
molecules associated with the ssDNA extension in the
(pol
)5 binding mode. Therefore, the value of
Fmax =
FDS + 3
F5 provides
F5 = 1.3 ± 0.1. Finally, there are two remaining parameters that must
be determined, K5 and
.
The solid lines in Fig. 3a are nonlinear
least-square fits of the experimental isotherms to Equation 11 which
provide K5 = (8 ± 3) × 105 M
1 and
= 5 ± 2. It should be pointed out that the value of K5 is similar to the K5 obtained for the DNA
substrate with the 3' ssDNA extension at the 3' end of the template
where the pol
can only bind in the (pol
)5 binding
mode (Table I). Such similarity reflects the fact that both binding
constants characterize a similar protein-nucleic acid binding process,
i.e. the formation of the (pol
)5 binding
mode. The value of
is in excellent agreement with previous studies
which showed that the cooperative interactions between the rat pol
molecules bound in the (pol
)5 binding mode to the ssDNA
polymer or the oligomer are very weak and characterized by
= 15 ± 6 (20). The binding isotherms in Fig. 3a were
also analyzed by letting four parameters float to within 20% of the determined values, e.g. KTP,
FTP,
FDS, and
KDS, and treating K5,
F5, and
as free fitting parameters.
Within experimental accuracy, this procedure returns the same values of
binding and spectroscopic parameters as the physically more intuitive
approach described above (data not shown).
Further examination of the effect of the dsDNA on the binding of rat
pol
to the template-primer DNA in the (pol
)16 and (pol
)5 binding modes has been performed using the DNA
substrate having the ssDNA extension 15 nucleotide residues in
length at the 3' end of the template (Fig. 1; substrate D). Notice,
with this substrate the polymerase cannot form the template-primer complex. The enzyme can bind the ssDNA extension only in either the
(pol
)16 or the (pol
)5 binding mode.
Fluorescence titrations of the DNA substrate, with rat pol
at two
different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C)
containing 100 mM NaCl, are shown in Fig.
3c. The relative increase of
the nucleic acid fluorescence reaches the value of 4.1 ± 0.2 at
saturation. The dependence of the relative fluorescence increase, as a
function of the average degree of binding,
vi, is
shown in Fig. 3d. There are two binding phases with
different affinities. In the high affinity phase, the degree of binding
reaches the value of 1 ± 0.2, indicating binding of a single
molecule of the enzyme. Extrapolation of
vi in
the low affinity binding phase to the maximum fluorescence increase,
Fmax = 4.1 ± 0.2, provides a value of
vi = 4.3 ± 0.3. Thus, at maximum
saturation, four pol
molecules can bind to the DNA substrate with
the ssDNA extension located at the 3' end of the template. The number
of bound pol
molecules is the same as observed for the DNA
substrate with the ssDNA extension at the 5' end of the template (Fig.
3b; Table II).

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Fig. 4.
Gapped DNA substrates that are used to
examine interactions of rat pol with gapped
DNA. The DNA substrates have two dsDNA parts, at the 5' end
(downstream from the primer) and the 3' end (primer location) of the
template strand, which are identical in all substrates. The dsDNA parts
are separated by the ssDNA of the gap containing 5 (substrate A), 4 (substrate B), 3 (substrate C), 2 (substrate D), and 1 (substrate E)
nucleotide residues. Substrate F is the same as A but contains a 5'
terminal phosphate group on the oligonucleotide in the dsDNA part at
the 3' end of the template strand, downstream from the primer.
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|
The obtained pattern of binding and the high stoichiometry can easily
be understood as a consequence of the existence of the two ssDNA enzyme
binding modes (see above; Ref. 20). At low enzyme concentrations, a
single molecule of rat pol
associates with the ssDNA. However, with
the ssDNA extension located at the 3' end of the template, the enzyme
cannot form a template-primer complex. In other words, only binding in
the (pol
)16 binding mode can occur at a low enzyme
concentration, which corresponds to the high affinity step (Fig.
3d). As the polymerase concentration increases, the (pol
)16 complex is replaced by the (pol
)5
binding mode with three enzyme molecules associated with the ssDNA
extension. At the same time, independent association of one polymerase
molecule with the dsDNA part of the DNA substrate occurs providing the final maximum stoichiometry of four rat pol
molecules bound to the DNA.
The complexity of this binding system is similar to the complexity of
the previously analyzed system of rat pol
binding to the DNA
substrate with the 5' ssDNA extension (Fig. 3, a and b). The only difference is the nature of the high affinity
complex. Instead of the template-primer complex, characterized by the
binding constant, KTP, the (pol
)16 binding mode is formed with the affinity characterized by the binding constant, K16.
Therefore, the same model, as described by Equations 7-11, applies to
the isotherms in Fig. 3c, with KTP
and
FTP replaced by
K16 and
F16, respectively. Also,
due to the large number of interaction and spectroscopic parameters,
the extraction of these parameters follows the same strategy as
outlined above. The binding of rat pol
in the (pol
)16 binding mode is significantly separated from the
binding to the dsDNA and the (pol
)5 binding mode, with
respect to the protein concentration. This separation allows us to
independently determine F16 as the slope,
F16 = 
F/
(
vi), of the initial part
of the plot in Fig. 3d which provides
F16 = 1.1 ± 0.1. The value of
K16 can be determined from the dependence of
vi as a function of the protein concentration
(plot not shown) which provides K16 = (5 ± 2) × 107 M
1. The dsDNA part
of the substrate is the same as other studied substrates (Table I).
Therefore, association of rat pol
with the dsDNA-binding site is
described by the same parameters, KDS = 2 × 106 M
1 and
FDS = 0.6. The final complex, at saturation,
contains one pol
molecule bound to the dsDNA and three pol
molecules bound in the (pol
)5 binding mode. Thus, the
value of
Fmax =
FDS + 3
F5 provides
F5 = 1.2 ± 0.1. Finally, there are only two remaining parameters that
must be determined, K5 and
.
The solid lines in Fig. 3c are nonlinear
least-square fits, using Equations 7-11 which provide
K5 = (5 ± 2) × 105
M
1 and
= 4 ± 2, respectively
(Table II). Thus, the formation of the (pol
)5 binding
mode is characterized by virtually the same interaction parameters as
that of the DNA substrate with the 5' ssDNA extension. Notice, the
obtained value, K16 = (5 ± 2) × 107 M
1, is by factors of ~125
and ~250 higher than the intrinsic binding constant of the (pol
)16 binding mode formed with the polymer and oligomer
ssDNAs, indicating that the presence of the dsDNA affects the affinity
of the (pol
)16 binding mode to an even greater extent
than that previously determined for the (pol
)5 binding
mode (Tables I and II) (20). Such a significantly higher value of
K16 strongly indicates that, when the enzyme is
bound in the (pol
)16 binding mode to the considered
substrate, the small 8-kDa domain interacts with the dsDNA providing an
additional contribution to the free energy of binding (see
"Discussion").
Binding of Rat Pol
to the Gapped DNA Substrates--
The
experiments and analyses described above focused on an effect of the
presence of the primer, or the dsDNA downstream from the primer, on rat
pol
interactions with the ssDNA extension in the (pol
)16 or (pol
)5 binding mode. In the
base-excision repair processes, one of the physiological substrates of
rat pol
is a gapped DNA which has a stretch of ssDNA embedded
between the primer and the dsDNA, downstream from the primer (7, 21, 22).
The gapped DNA substrates, used to examine interactions with rat pol
, are depicted in Fig. 4. All DNA substrates contain two dsDNA parts
each having 10 bp. The primary structure of the dsDNA parts is
identical in all gapped DNAs and is the same as analogous dsDNA
fragments in the previously analyzed DNA substrates (Figs. 1;
substrates A, B, C, and D). The dsDNA parts are separated by a ssDNA
gap having five, four, three, two, and one nucleotide residues. The
nucleotide residues in the ssDNA gap are all ethenoadenosines (
A)
which provide the signal to monitor the binding.
Fluorescence titrations of the DNA substrate, having a ssDNA gap of
five nucleotide residues in length (Fig. 4; substrate A), with rat pol
at two different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, are shown in Fig.
5a. The relative nucleic acid
fluorescence increase reaches the value of 7.2 ± 0.3 at
saturation. Fig. 5b shows the dependence of the observed
relative fluorescence increase as a function of the average degree of
binding,
vi, of the enzyme. The plot shows
nonlinear behavior indicating two binding phases. In the high affinity
phase, the degree of binding reaches the value of 1 ± 0.2, thus,
a single molecule of the polymerase binds in this phase. However, the
relative fluorescence increase, as determined from the initial slope,

F/
(
vi), of the plot is only 1.8 ± 0.1, very similar to the values obtained for the DNA substrates having
ssDNA extensions with 15 nucleotide residues (Tables I and II). Most of
the fluorescence increase comes from the additional binding of pol
molecules to the DNA. Extrapolation of the low affinity phase to the
maximum fluorescence increase,
Fmax = 7.2 ± 0.3 provides a value of
vi = 3.3 ± 0.3. Thus, the data indicate that, at saturation, three rat pol
molecules bind to the considered gapped DNA substrate.

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Fig. 5.
a, fluorescence titrations
( ex = 325 nm, em = 410 nm) of the gapped
DNA substrate (Fig. 4; substrate A), with the ssDNA gap having 5 nucleotide residues, with rat pol in buffer C (pH 7.0, 10 °C),
containing 100 mM NaCl, at 2 different concentrations of
the nucleic acid: , 4.5 × 10 7 M;
( ) 1.5 × 10 6 M. The solid
lines are nonlinear least-square fits of the fluorescence
titration curves according to the three binding-site model. The
lines are plotted using a single set of parameters:
KG = 5 × 107
M 1,
KDS1 = 2 × 106 M 1,
KDS2 = 3 × 105 M 1, FG = 1.8, FDS1 = 0.6, and
FDS2 = 5 (Table III;
details in text). b, the dependence of the relative
fluorescence increase, F, upon the degree of binding,
vi, of the rat pol -gapped DNA complex. The
values of the degree of binding have been determined using the
quantitative method described under "Experimental Procedures." The
solid lines are the limiting slopes of the two binding
phases. The dashed line is an extrapolation of the degree of
binding to the maximum value of the observed fluorescence increase
Fmax = 7.2 ± 0.2 that provides the
maximum stoichiometry of 3.3 ± 0.3 of the rat pol -gapped DNA
complex. c, fluorescence titrations the gapped DNA substrate
(Fig. 4; substrate E) having the ssDNA gap with 1 nucleotide residue,
with rat pol in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, at two different concentrations of the nucleic
acid: , 8.8 × 10 7 M; , 2.22 × 10 6 M. The solid lines are
nonlinear least-square fits of the fluorescence titration curves
according to the three binding-site model (Table III; details in text).
d, the dependence of the relative fluorescence increase,
F, upon the degree of binding, vi,
of the rat pol -gapped DNA complex (Fig. 4; substrate E). The
dashed line is an extrapolation of the degree of binding to
the maximum value of the observed fluorescence increase
Fmax = 2.1 ± 0.2 that provides the
maximum stoichiometry of 3.1 ± 0.3.
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|
The rat pol
affinity for the dsDNA is much lower than the affinity
observed for the first binding phase (Tables I and II), therefore, the
high affinity phase must correspond to the formation of a complex that
includes the ssDNA gap. The low affinity phase must then correspond to
the association of the remaining two pol
molecules with the two
dsDNA parts of the substrate. Binding to the dsDNA, downstream from the
primer, is most probably not affected by the enzyme molecule associated
with the gap. However, this may not be the case with the binding to the
dsDNA part at the primer location. Due to the limited size of the gap,
the enzyme bound to the gap may be forced to invade the duplex DNA to a
larger extent than in the previously examined substrates with ssDNA
extensions where the large 31-kDa domain partially engages the
ssDNA in the complex (see "Discussion"). Thus, in general, two
dsDNA parts of the gapped DNA substrate may not be equivalent. In the
simplest approach, the association of rat pol
with the gapped DNA
can be treated as ligand binding to three distinct binding sites. The
total partition function, ZG, of the system is
defined by,
|
(Eq. 12)
|
where KG is the binding constant characterizing
the association with the ssDNA gap.
KDS1 and
KDS2 are the binding constants
characterizing the association with the dsDNA downstream from the
primer and at the primer location, respectively. The degree of binding,
vi, is then defined as,
|
(Eq. 13)
|
The observed relative fluorescence change,
F, is
described by,
|
(Eq. 14)
|
where
|
(Eq. 15)
|
|
(Eq. 16)
|
|
(Eq. 17)
|
There are six independent parameters in Equations 12-17. As we
pointed out above, such a large number of parameters precludes their
determination in a single, fitting procedure. The approach to extract
them is similar to the one already described for the other DNA
substrates (see above). Much higher affinity of the enzyme to the gap
region than the dsDNA parts of the nucleic acid allows us to determine
FG as the slope, FG = 
F/
(
vi), of the initial part
of the plot in Fig. 5b, which provides
FG = 1.8 ± 0.1. The value of
KG can be determined from the analysis of the
dependence of
vi, as a function of the protein
concentration in the low protein concentration range (plot not shown),
which provides KG = (5 ± 2) × 107 M
1. Association of rat pol
with the dsDNA, downstream from the primer, is characterized by the
same parameters,
KDS1 = (2 ± 0.5) × 106 M
1
and
FDS1 = 0.6 ± 0.1, as determined for this binding site for other studied substrates
(Tables I and II). At saturation, the complex contains one rat pol
bound to the gap and two enzyme molecules associated with each of the
dsDNA parts of the substrate. Therefore, the value of
Fmax =
FDS1 +
FDS2 +
FG which provides
FDS2 = 5 ± 0.1. Thus, there is only one remaining parameter that has to be determined,
KDS2. The solid
lines in Fig. 5a are nonlinear least-square
fits of the experimental isotherm to Equations 12-17 with
KDS2 = (3 ± 1) × 105 M
1. The theoretical curves
provide an excellent description of the experimental titration curves.
Alternatively, knowing
FG,
FDS1,
FDS2, and
KDS1, or letting these
parameters float to within 20% of the estimated values, one can fit
the experimental curves with two parameters KG and
KDS2 (data not shown).
Within experimental accuracy, these procedures return very similar
values of all binding parameters as the more intuitive approach
described above.
The obtained value of KG for the ssDNA gap of 5 nucleotide residues is a factor of ~2000 higher than the intrinsic binding constant, K5 = 2 × 104
M
1, characterizing the formations of only the
(pol
)5 binding mode formed exclusively on the ssDNA
oligomer (20). The determined affinity of the enzyme for the gap is by
factors of ~160 and ~100 higher than the affinities for the DNA
substrates with the ssDNA extension capable of accommodating the enzyme
only in the (pol
)5 binding mode (Tables I). Also, the
value of KG is by a factor of ~25 higher than the
value of KDS1
characterizing the independent binding to the dsDNA (Tables I and III).
It is evident that such a large difference in affinity provides the enzyme with a significant preference for the gapped DNA, as compared with the ss and dsDNAs.
Examination of rat pol
binding to gapped DNA substrates, having
ssDNA gaps of different sizes (Fig. 4), addresses the very important
question: how is the high affinity and stoichiometry for the enzyme-gap
complex affected by the size of the ssDNA gap? The smallest possible
ssDNA gap is 1 nucleotide in length. Fluorescence titrations of the DNA
substrate having only 1 nucleotide residue in the ssDNA gap, with rat
pol
, at two different nucleic acid concentrations, in buffer C (pH
7.0, 10 °C), containing 100 mM NaCl, are shown in Fig.
5c. The relative nucleic acid fluorescence increase reaches
the value of 2.1 ± 0.2 at saturation, which is much lower than
the maximum fluorescence increase observed for the gapped DNA substrate
with 5 nucleotide residues in the ssDNA gap (Fig. 5a; see
"Discussion"). Fig. 5d shows the dependence of the
observed relative fluorescence increase as a function of the average
degree of binding,
vi, of the enzyme on the
substrate. There are clearly two binding phases. In the high affinity
phase, the degree of binding reaches the value of 1 ± 0.2, indicating the binding of a single molecule of the polymerase. The
relative fluorescence increase accompanying the binding in this step is

F/
(
vi) = 0.9 ± 0.1. Extrapolation of the low affinity phase to the maximum
fluorescence increase,
Fmax = 2.1 ± 0.2 provides a value of
vi = 3.1 ± 0.3. Thus, despite the fact that the considered substrate has only a single nucleotide residue in the ssDNA gap, at maximum saturation three rat
pol
molecules bind to the substrate.
Titration curves in Fig. 5c have been analyzed as already
described for the gapped DNA substrate with 5 nucleotide residues in
the ssDNA gap, using the three-site binding model as described by
Equations 12-17. The solid lines in Fig. 5c are
nonlinear least-square fit of the experimental isotherms with
KG = (2 ± 1) × 107
M
1,
FG = 0.9 ± 0.1, KDS1 = (2 ± 0.5) × 106 M
1,
FDS1 = 0.6 ± 0.1, KDS2 = (2.5 ± 0.8) × 105 M
1, and
FDS2 = 0.6 ± 0.1. The interaction and spectroscopic parameters for all studied
gapped DNA substrates are included in Table
III. Thus, the size of the ssDNA gap does
not affect the enzyme affinity for the ssDNA gap, as reflected in
virtually the same values of KG for all examined
substrates. On the other hand, there are differences between different
gapped DNAs, with respect to the values of the maximum fluorescence
increase,
Fmax, and spectroscopic parameters,
FG and
FDS2. These
differences suggest that, despite very similar affinities, the
structures of the ssDNA in formed complexes are different (see
"Discussion").
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Table III
Thermodynamic and spectroscopic parameters for rat pol binding to
gapped DNA substrates with a different number of nucleotide residues in
the ssDNA gap (Fig. 4; substrates A, B, C, D, E, and F) in buffer C (pH
7.0, 10 °C) containing 100 mM NaCl
The errors are standard deviations determined using three to four
independent titrations.
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|
Effect of the 5' Terminal Phosphate Downstream from the Primer on
the Rat Pol
Association with the Template-Primer and Gapped-DNA
Substrates--
Steady-state kinetic studies of rat pol
activities
on the template-primer and gapped DNA substrates clearly indicate that the presence of the 5' terminal phosphate group, downstream from the
primer, plays an important role in DNA substrate recognition and
catalysis (21-23). However, direct thermodynamic analysis of the
effect of the 5' terminal phosphate on the binding of rat pol
to
DNA substrates has never been addressed before. To elucidate the role
of the 5' terminal phosphate group in the energetics of the
polymerase interactions with the DNA, we first examined the interaction
of rat pol
with the DNA substrate having a 5' terminal phosphate on
the oligonucleotide forming the dsDNA at the 5' end of the template
strand (Fig. 1; substrate E). The ssDNA extension having 15 nucleotide
residues is located at the 3' end of the template strand. Therefore,
the enzyme can bind the ssDNA extension in both the (pol
)16 and (pol
)5 binding modes.
Fluorescence titrations of the DNA substrate with rat pol
at two
different nucleic acid concentrations, in buffer C (pH 7.0, 10 °C),
containing 100 mM NaCl, are shown in Fig.
6a. The relative maximum
nucleic acid fluorescence increase reaches the value of
Fmax = 3.4 ± 0.2, at saturation, which
is significantly lower than
Fmax = 4.4 ± 0.2 obtained for the DNA substrate without the phosphate group
(Table II). Thus, the data show that the presence of the 5' terminal
phosphate affects the extent of the structural changes in the ssDNA
induced by the enzyme in the complex. At this point, it should be
mentioned that in the presence of Mg2+, pol
catalyzes a
slow release of the 5' terminal deoxyribose phosphate (40). Therefore,
we also examined the binding of the enzyme to the considered DNA
substrate and the gapped DNA discussed below, in the absence of
MgCl2 (data not shown). Both sets of data, in the presence
and absence of Mg2+, give, within experimental accuracy,
very similar results. Thus, in our solution conditions (low
Mg2+ and low temperature) the catalysis is very slow, on
the time scale of the titration experiments and does not affect the
studied equilibrium.

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Fig. 6.
a, fluorescence titrations
( ex = 325 nm, em = 410 nm) of the
template-primer DNA substrate (Fig. 1; substrate E), containing
the ssDNA extension with 15 nucleotide residues at the 3' end of
the template strand, and the 5' terminal phosphate on the nucleotide
complementary to the template strand, with rat pol in buffer C (pH
7.0, 10 °C), containing 100 mM NaCl, at two different
concentrations of the nucleic acid: , 4.5 × 10 7
M; , 1.5 × 10 6 M. The
solid lines are nonlinear least-square fits of the
fluorescence titration curves according to the statistical
thermodynamic model defined by Equations 7-11. The model takes into
account the overlap of potential binding sites and cooperative
interactions in the formation of the (pol )5 binding
mode on the ssDNA extension, formation of the (pol )16
binding mode, and binding of the enzyme to dsDNA part of the DNA
substrate (details in text). The theoretical lines are plotted using a
single set of parameters (Table II). b, the dependence of
the relative fluorescence increase, F, upon the average
degree of binding, vi, of the rat pol -DNA
substrate complex. The dashed line is an extrapolation of
the degree of binding to the maximum value of the observed fluorescence
increase Fmax = 3.4 ± 0.2 which
provides the maximum stoichiometry of 4.3 ± 0.3.
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|
The dependence of the observed
F as a function of the
average degree of binding,
vi, of rat pol
on
DNA, is shown in Fig. 6b. In the high affinity phase, the
degree of binding reaches the value of 1 ± 0.2, indicating
binding of a single molecule of the enzyme. Extrapolation of
vi to the maximum fluorescence increase,
Fmax = 3.4 ± 0.2, provides a value of
vi = 4.3 ± 0.3. Clearly, the presence of
the 5' terminal phosphate does not affect the stoichiometry of the
complex. The slope,
F = 
F/
(
vi) =
F16, of the initial part of the plot in Fig.
6b provides
F16 = 1.5 ± 0.1. This value is slightly higher than the one obtained for the DNA
substrate without the terminal phosphate (Table II), indicating
different ssDNA structures in both complexes.
The analysis of the titration curves in Fig. 6a has been
performed in the same way as described for the DNA substrate having the
ssDNA extension with 15 nucleotide residues at the 3' end of the
template strand (Figs. 3, c and d). The
solid lines in Fig. 6a are nonlinear least square
fits of the experimental isotherms with K16 = (7 ± 3) × 107 M
1,
F16 = 1.5 ± 0.1, KDS = (2 ± 0.5) × 106 M
1,
FDS = 0.6 ± 0.1, K5 = (9 ± 2) × 105
M
1,
= 3 ± 1, and
F5 = 0.94 ± 0.06 (Table II). Thus, the
presence of the 5' terminal phosphate has little effect on the values
of all spectroscopic and interaction parameters, as compared with the
same DNA substrate without the phosphate group (Fig. 1; Table II).
Analogous fluorescence titrations have been performed using the DNA
substrate with the ssDNA gap having 5 nucleotide residues and the 5'
terminal phosphate (Fig. 4; substrate F), with rat pol
(data not
shown). In the high affinity phase,
vi reaches
the value of 1 ± 0.2, indicating binding of a single enzyme molecule. Extrapolation of
vi to the maximum
fluorescence increase,
Fmax = 6.7 ± 0.2, provides a value of
vi = 3.2 ± 0.3, indicating that the maximum stoichiometry of three pol
molecules
bound to the DNA is not affected by the presence of the 5' terminal
phosphate. The relative fluorescence increase reaches the value of
6.7 ± 0.2, at saturation, which is similar to the same parameter
obtained for the DNA substrate without the phosphate group (Table III).
However, the slope,
F = 
F/
(
vi) =
FG,
of the initial part of the titration curve provides
FG = 3 ± 0.1. This value is significantly
higher than the
FG = 1.8 ± 0.1 obtained for
the DNA substrate without the terminal phosphate (Table III). Because
FG characterizes the conformational state of the
ssDNA of the gap, in the complex with the enzyme molecule directly
bound to the gap, such a large value of
FG
indicates that the presence of the 5' terminal phosphate strongly
affects the conformational state of the ssDNA in the complex with the
polymerase (see "Discussion"). The nonlinear least-square fit of
the experimental isotherms, using Equations 12-17 provide
KG = (5 ± 2) × 107
M
1,
FG = 3 ± 0.1, KDS1 = (2 ± 0.5) × 106 M
1,
FDS1 = 0.6 ± 0.1, KDS2 = (2.1 ± 0.8) × 105 M
1, and
FDS2 = 3.1 ± 0.1. Thus, despite the structural differences, as expressed by the
much larger
FG, the presence of the 5' terminal
phosphate does not significantly affect the energetics of rat pol
binding to the gapped DNA (Table III).
 |
DISCUSSION |
Despite simplified catalytic activities, the recognition process
of the template-primer and gapped DNA substrates by rat pol
is a
complex process, as indicated by the direct thermodynamic studies
described in this work. Such complex behavior was already evident in
previous studies of the binding of the enzyme exclusively to the ssDNA
(20). The polymerase binds the ssDNA using two different binding modes,
which have a different number of occluded nucleotide residues in the
protein-ssDNA complex. These findings were possible due to the
application of the quantitative fluorescence titration technique that
allowed us to determine the average degree of binding,
vi, of the protein-DNA complexes, without assumptions about the relationship between the observed signal used to
monitor the binding and the stoichiometry of the studied complexes
(32-35). Moreover, the approach allows us to extract spectroscopic parameters characterizing the structural changes of the
nucleic acid accompanying the formation of the particular complexes.
The Stoichiometry of Rat Pol
-Template-Primer DNA Substrate
Complexes is Higher Than Predicted by the Crystal Structure of the
Enzyme-DNA Complex--
A stoichiometry of the ligand-macromolecule
complex, at saturation, is a model-independent parameter. This quantity
defines the maximum number of ligand molecules that can bind to the
macromolecule. A surprising feature of the examined interactions
between the template-primer DNA substrates and rat pol
is the high
stoichiometry of the formed complexes at saturation. Thus, four rat pol
molecules are bound to the DNA when the ssDNA extension has a
length of 15 nucleotides. Even with the template-primer substrates,
which have a ssDNA extension with only 5 nucleotide residues (Fig. 1; substrates A and B), two enzyme molecules associate with the DNA. These stoichiometries, determined in solution, are very different from
the reported co-crystal structure of the similar enzyme-template-primer complexes, although, more recent extensive, crystallographic studies revealed the existence of more than one pol
molecule bound to the
template-primer DNA (16, 17).
The observed stoichiometries can be understood in the context of the
known enzyme affinity for the dsDNA conformation and the existence of
the two ssDNA binding modes (20). Moreover, the analysis of the formed
complexes is facilitated by early studies showing that the polymerase
has two distinct DNA-binding domains possessing different preferences
for the two nucleic acid conformations (14-19). The site with the
preference for the ssDNA is located on the small 8-kDa domain, while
the site with the preference for the dsDNA is located on the large
31-kDa catalytic domain (14, 18, 19).
The (pol
)16 binding mode, in which the enzyme occludes
16 ± 2 nucleotide residues, is formed in the large excess of the ssDNA (20). In this mode, both domains of the enzyme are engaged in
interactions with the nucleic acid. The transition to the (pol
)5 binding mode is induced by the increased protein
concentration resulting from the increase of the protein binding
density on the DNA. Therefore, when the availability of the ssDNA is
decreasing, the protein, initially bound to 16 nucleotide residues, is
forced to bind the ssDNA with a lower 5-nucleotide site-size, forming the (pol
)5 binding mode, with the 8-kDa domain
predominantly interacting with the DNA (20). The low availability of
the ssDNA is already built into the structure of the template-primer
DNA substrate, having the 3' ssDNA extension with only 5 nucleotides. The extension can only accept one enzyme molecule in the (pol
)5 binding mode. The second enzyme molecule must bind to
the 10-bp long dsDNA part of the substrate using the dsDNA-binding site
located on the large 31-kDa domain (16, 17).
At low protein concentrations the template-primer substrate, having the
3' ssDNA extension with 15 nucleotide residues, can initially accept a
single pol
molecule only in the (pol
)16 binding
mode. However, as the protein concentration increases, this mode is
being replaced by the (pol
)5 binding mode leading to
three enzyme molecules bound to the ssDNA extension, at saturation. Binding of the single rat pol
molecule to the dsDNA part of the
substrate results in 4 enzyme molecules associated with the DNA.
For the template-primer substrates having the ssDNA extensions at the
5' end of the substrate, the situation is different (Fig. 1). At low
protein concentrations, the extension with 15 nucleotide residues can
accept the enzyme in the (pol
)16 or the (pol
)5 binding mode. However, because the ssDNA extension is
located at the 5' end of the substrate, the enzyme forms a template-primer complex instead of the (pol
)16 binding
mode, where the 8-kDa domain binds the ssDNA of the template and the large 31-kDa domain is engaged in interactions with the ss-ds DNA
junction (16). The formation of such a complex is reflected in the
different interaction and spectroscopic parameters, as compared with
the DNA substrate having the ssDNA extensions with 15 nucleotide
residues at the 3' end of the substrates where only the (pol
)16 binding mode can be formed (Table II) (20). Also, a
lower fluorescence change and a much higher affinity characterizing the
binding of the enzyme to the DNA substrate with the ssDNA extension
having 5 nucleotide residues indicate that, instead of the (pol
)5 binding mode, the polymerase binds the ss-ds DNA junction of the DNA substrate using its 31-kDa domain (16, 20).
Notice the maximum stoichiometries are not affected by either the
existence of the template-primer complex or the binding to the ss-ds
DNA junction. Moreover, the binding constants of the second pol
molecule are unaffected by the template-primer complex, indicating that
the 10-bp long dsDNA is fully available to the enzyme (Table I). Thus,
these data also suggest that in the template-primer complex, or when
bound to the ss-ds DNA junction, contrary to the crystal structure of
the complex, the polymerase must encompass less than 7 bp of the dsDNA
of the substrate (16, 17).
The 8-kDa Domain Interacts with the dsDNA, Downstream from the
Primer, in Both the (pol
)5 and (pol
)16
Binding Modes--
Experiments with DNA substrates having the ssDNA
extension at the 3' end of the substrate provide strong evidence that
the 8-kDa domain of rat pol
is involved in interactions with the dsDNA located at the 5' end of the substrate. The intrinsic binding constants for the formation of the (pol
)5
and (pol
)16 binding modes, exclusively with the
ssDNA oligomer d
A(p
A)15, in the same solution
conditions as applied in this work, are K5 = (2 ± 1) × 104 M
1
and K16 = (2 ± 1) × 105 M
1, respectively (20).
However, the values of the corresponding constants determined for the
template-primer substrates having the 3' ssDNA extension, where the
enzyme can only form either the (pol
)5 or the (pol
)16 binding mode, are K5 = (7 ± 2) × 105 M
1 and
K16 = (5 ± 2) × 107
M
1, respectively (Table II). Thus, in the
presence of the dsDNA, downstream from the primer, the affinities of
both binding modes are increased by factors of ~35 and ~250,
respectively. These results strongly indicate that in both binding
modes the 8-kDa domain is engaged in interactions with the dsDNA of the
DNA substrate. On the other hand, there is a significant difference
between the effects of the dsDNA on the two binding modes. A stronger
effect is observed for the (pol
)16 binding mode,
suggesting that in this binding mode the 8-kDa domain has more
flexibility to engage the dsDNA. This conclusion is supported by the
observed lower fluorescence changes accompanying the formation of the
(pol
)16 binding mode, indicating a more flexible
structure at the interface of the protein-nucleic acid complex (20).
Such flexibility is limited in the (pol
)5 binding mode
where the enzyme predominantly interacts with the ssDNA through the
8-kDa domain, leading to a much stronger immobilization of the DNA and
the domain in the complex (20).
Energetics of the Rat Pol
-Template-Primer Complex--
As we
discussed above, when the ssDNA extension with 15 nucleotide residues
is located at the 5' end of the DNA substrate (Fig. 1; substrate C),
rat pol
can form a template-primer complex (16, 17). In this
complex the 8-kDa domain of the enzyme interacts with the ssDNA of the
template, while the catalytic 31-kDa domain is engaged in interactions
with the ss-ds DNA junction (16). The binding constant characterizing
the formation of the template-primer complex
KTP = (4 ± 2) × 107
M
1 is by a factor of ~2000 higher than the
intrinsic binding constant determined for the (pol
)5
binding mode (K5 = (2 ± 1) × 104 M
1), where the enzyme
exclusively interacts with the ssDNA using the 8-kDa domain, and by a
factor of ~10 higher than the binding constant KJ = (4.6 ± 0.6) × 106
M
1, characterizing the independent
interactions of the polymerase with the ss-ds DNA junction, through the
large 31-kDa domain (Tables I and II).
Comparison of these binding constants provides the first indication
that the association of the enzyme with the template-primer DNA is not
composed of the independent interactions of the two domains of the
enzyme with two different conformations of the DNA substrate. The free
energy change accompanying the formation of the template-primer complex
is
G
= RT
lnKTP, where R is the gas constant
and T is the temperature in Kelvin degrees (41). Introducing
the value of KTP provides
G
=
9.9 ± 0.2 kcal/mol. Analogous calculation for the (pol
)5 binding
mode and the ss-ds DNA junction result in
G
=
5.5 ± 0.3 and
G
8.6 ± 0.2 kcal/mol, respectively. Thus, the absolute value of free energy of the
template-primer complex formation is significantly lower than the
absolute value of the sum of independent free energies of binding
(14.1 ± 0.5 kcal/mol) exclusively in the (pol
)5
binding mode and to the ss-ds DNA junction.
The free energy cost of the template-primer complex formation can be
approximately obtained using a general approach introduced by Jencks
(42). The free energy of simultaneous binding of the ssDNA and the
ss-ds DNA junction, as parts of the template-primer DNA, can be defined
as,
|
(Eq. 18)
|
where
G
is the
"connection" free energy change representing a gain or loss of free
energy as a result of the simultaneous binding of the ss- and dsDNAs as
structural parts of the larger substrate, the template-primer DNA.
Introducing the values of
G
,
G
, and
G
into Equation 10 provides
G
= 4.2 ± 0.7 kcal/mol.
Notice that the value of
G
is positive, thus, the complex of the enzyme with the template-primer
substrate has a significant excess of the free energy gained at the
expense of the free energy of binding of the ssDNA and the ss-ds DNA
junction of the substrate. At this point, it is not known whether or
not the excess free energy is accumulated in the polymerase or the DNA
or both. Elegant crystallographic studies indicate large conformational changes in both pol
and the DNA substrate in the enzyme-DNA complex, as compared with the structures of both macromolecules in the
absence of interactions (16). Such excess free energy can be released
at different stages of enzyme action whenever interactions with one of
the DNA conformations are weakened. For instance, the free energy
release can play an important role in the functioning of the enzyme,
particularly in the mechanical translocation, where the affinities of
the enzyme domains for different conformations of the DNA substrate
transiently change (1, 16, 17, 43). Because of similarities of the
enzyme structure and its complex with the template-primer DNA among
different polymerases, it is very possible that a similar pattern of
free energy changes, i.e. accumulation of
G°
at the expense of the free energy of binding accompanies interactions
of other DNA polymerases with a template-primer DNA.
The Size of the ssDNA Gap Has Little Effect on the Stoichiometry
and Affinity of the Rat Pol
Complexes with Different Gapped DNA
Substrates--
The maximum stoichiometry of the enzyme-DNA complex
and the intrinsic affinities of the bound pol
molecules, to various gapped DNA substrates examined in this work, are not affected by the
diminishing size of the ssDNA gap (Table III). Thus, the same
stoichiometry of the complex is preserved despite the fact that the
largest and the smallest gaps differ by as much as 4 nucleotide
residues that are within the site-size of 5 ± 2 of the (pol
)5 binding mode (20). It is still very possible that the
formation of the (pol
)5 binding mode is a part of the
gap recognition mechanism, particularly for the larger gaps. Whether or
not the (pol
)5 binding mode is a part of the
recognition mechanism for large ssDNA gaps can only be resolved by
kinetic methods. However, the obtained results indicate that the final equilibrium complex with the gap is very different from the (pol
)5 binding mode.
Steady-state kinetic data have clearly shown that the polymerase can
processively catalyze the DNA synthesis on all DNA substrates, analogous to the ones used in this work (21, 22). Thus, the active site
of the enzyme, located on the 31-kDa catalytic domain, must always be
placed close to the 3' end of the primer oligomer (16). In other words,
in all examined gapped DNA substrates, the 31-kDa domain interacts with
the ss-ds DNA junction in the same orientation, with respect to the
dsDNA. Therefore, what has to change, to preserve the same
stoichiometry and the same intrinsic affinities of all three bound pol
molecules as the size of the gap diminishes, is the location of the
8-kDa domain of the enzyme molecule bound to the gap, with respect to
its large domain and the gap. The independence of the high intrinsic
affinity of the enzyme molecule, strictly bound to the ssDNA gap, upon
the size of the gap, indicates that in the complexes with different
gapped DNAs, the enzyme forms all crucial contacts with the DNA
substrate. Studies of rat pol
interactions with the template-primer
DNA substrates, described above, clearly indicate that the small 8-kDa domain of the enzyme can engage in interactions with the dsDNA (see
above). This result is of paramount importance because the dsDNA is the
only nucleic acid conformation available to the 8-kDa domain in the
complexes with small gaps. The unaffected affinity would reflect the
fact that, independently of the size of the gap of the examined DNA
substrates, both domains are interacting with the same conformation of
the DNA. The 31-kDa domain is bound to the junction of the ss- and
dsDNA, while the 8-kDa domain is bound to the dsDNA, downstream from
the primer.
Model for the Gapped-DNA Substrate Recognition by Rat Pol
--
The ssDNA affinity of the 8-kDa domain has been invoked as a
major factor in the recognition of gapped-DNA substrates (22). Although, as we stated above, this may still be true, particularly for
the larger gaps, the recognition of the small gaps cannot rely on such
ssDNA affinity because there is not enough ssDNA available for binding
(20). We propose the following plausible mechanism of the small ssDNA
gap recognition by rat pol
on the bases of the results and analyses
obtained in this work.
Rat pol
binds the dsDNA, as well as the ss-ds DNA junction of the
substrate, using its large catalytic 31-kDa domain. Binding to the
ss-ds DNA junction, in the absence of the downstream dsDNA part, shows
only a slight, if any, preference over the dsDNA (Tables I and II).
However, only when the polymerase is associated with the ss-ds DNA
junction does the enzyme undergo an allosteric conformational transition that changes the orientation of the small 8-kDa domain, with
respect to the 31-kDa domain and the gap. Binding of the enzyme to the
dsDNA alone does not induce this allosteric transition. In the acquired
conformation, the small 8-kDa domain approaches and binds the dsDNA,
downstream from the primer. As a result, the affinity of the enzyme for
the gap is increased by a factor of ~25 over the affinity for the
dsDNA surrounding the gap and providing the enzyme with a significant
preference for the ssDNA gap over the surrounding dsDNA (Tables I-III).
Schematic structures of rat pol
complexes with the dsDNA and
gapped DNA substrates, according to the proposed model, are
shown in Fig. 7.

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Fig. 7.
Schematic representation of two different
complexes of rat pol with the dsDNA
(A) and with the gapped DNA substrate
(B), based on the results obtained in this work.
The enzyme binds both substrates using its large 31-kDa domain.
However, in the complex with the gapped DNA, interactions between the
31-kDa domain (gray area) and the ss-ds DNA junction of the
DNA substrate induce a specific allosteric transition of the enzyme. As
a result, the small 8-kDa domain (black area), which has an
intrinsic affinity for the dsDNA, is at a shorter distance from the DNA
and binds the dsDNA conformation downstream from the primer. These
additional interactions lead to the significantly amplified affinity
for the gapped DNA substrate. Binding exclusively to the dsDNA (A) does
not induce the conformational transition of the polymerase. As a
result, the enzyme interacts with the nucleic acid, using only the
affinity of its dsDNA-binding site located on the large 31-kDa domain.
The polymerase remains in its unchanged conformation with the 8-kDa
domain at a significant distance from the nucleic acid, and not able to
engage in interactions with the dsDNA.
|
|
Notice, in this model the selectivity of rat pol
for the gapped DNA
substrates is not generated by the high affinity of the enzyme for the
ssDNA, but by an allosteric conformational transition of the enzyme-DNA
complex, specific for the substrate structure bound to the 31-kDa
domain, the ss-ds DNA junction. The data and analyses discussed above,
as well as the results of our previous studies on rat pol
interactions with the ssDNA, indicate that the two functional and
structural domains of the enzyme form a complex allosteric system,
whose properties are crucial for the recognition of the physiological
substrate of the enzyme, the gapped DNA. The signal from one domain is
transferred to another domain allowing the enzyme structure to
specifically respond to the structure of the DNA in the complex and to
amplify the affinity.
The Presence of the 5' Terminal Phosphate, Downstream from the
Primer, Has Little Effect on the Affinities of Rat Pol
for
Template-Primer and Gapped DNA--
Steady-state kinetic data
indicated that the 5' terminal phosphate group, downsteam from the
primer, strongly affects the activities of rat pol
(21-23). Thus,
the distributive character of the DNA synthesis by the enzyme,
particularly on the gapped DNA substrates, is changed into a processive
one in the presence of the 5' terminal phosphate (21, 22). The simplest
explanation of this effect is that the phosphate group increases the
ground-state affinity of the enzyme for the DNA and, in turn, the
lifetime of the enzyme-DNA complex (21, 22). However, direct
thermodynamic studies, reported in this work, show that the
ground-state affinity of the enzyme for the DNA substrate with the 3'
ssDNA extension, as well as for the gapped DNA, is not significantly
affected by the presence of the 5' terminal phosphate group (Tables II
and III).
On the other hand, the presence of the phosphate group has a profound
effect on the structure of the ssDNA in the gap, when complexed with
the polymerase. The fluorescence change,
FG, directly accompanying the polymerase binding to the ssDNA gap having
the 5' terminal phosphate downstream from the primer is 3 ± 0.1, as compared with the
FG = 1.8 ± 0.1 determined in the absence of the PO
group (Table III). Because at the applied excitation wavelength
(
ex = 325 nm), only ethenoadenosine is excited, the
observed increase results from an increase of the quantum yield of the
nucleic acid in the complex with the polymerase. The fluorescence of
A is dramatically quenched (8-12-fold) in etheno-oligomers and
polymers as compared with the free AMP (44, 45). A dynamic
model, in which the motion of
A leads to quenching via
intramolecular collision, has been proposed as a predominant mechanism
of the observed strong quenching (44). Thus, the fluorescence of
A
in etheno-derivatives of the polymer and oligomer nucleic acids is
predominantly affected by the mobility and separation of the bases and
not by the polarity of the environment. Therefore, a large increase of
the
FG value indicates that, in the presence of
the 5' terminal phosphate group, the ssDNA of the gap assumes a much
more rigid conformation with significantly increased distances between
the bases.
It is rather certain that recognition and DNA synthesis on the
template-primer, or gapped DNA substrates, are complex, multistep processes that must include binding steps as well as chemical catalysis. In the context of the previous steady-state studies, our
results indicate that the presence of the 5' terminal phosphate group
has an effect on the functioning of the enzyme, not through increasing
the ground-state affinity, but by affecting some of the subsequent
chemical steps. Large differences between the ssDNA conformations in
the gap, in the absence and presence of the terminal phosphate, provide
the first structural clue as to how this can happen. The increased
rigidity of the template strand in the ssDNA gap, as well as the larger
separation of the bases, can facilitate the dNTP binding and
recognition. In turn, this will lead to the increased processivity of
the enzyme action.