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J. Biol. Chem., Vol. 277, Issue 23, 20316-20327, June 7, 2002

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Dynamics of Gapped DNA Recognition by Human Polymerase ^*

Maria J. Jezewska, Roberto Galletto, and Wlodzimierz Bujalowski

From the Department of Human Biological Chemistry and Genetics and The Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-0153

Received for publication, January 28, 2002, and in revised form, March 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Kinetics of human polymerase  binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ~600-1200 s¹. This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5'-terminal PO group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Polymerase  is one of a number of recognized DNA-directed polymerases of the eukaryotic nucleus (1-5). A characteristic feature of human pol ¹ is a "simplified" set of activities. The enzyme lacks intrinsic accessory activities, such as 3' or 5' exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1, 5-7). This limited repertoire of activities reflects the very specialized function of the enzyme in human cell repair machinery which includes the gap filling synthesis involved in mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA (1, 4, 5-12). Human pol  is a single polypeptide of ~39,000 kDa. The crystallographic structures of both rat and human pol  have been determined at 3.6- and 2.3-Å resolution (13-15). The feature that distinguishes the pol  structure from other polymerases is the presence of a small 8-kDa domain connected with the tip of the fingers through a tether of 14 amino acids (13, 16). The domain possesses the enzymatic ability to catalyze the release of the 5'-terminal deoxyribose phosphate residue from the incised apurinic-apirimidinic site that is a common intermediate product in base excision repair (12). The active site of the DNA synthesis resides predominantly in the large 31-kDa domain of the enzyme (13-15).

A puzzling problem in the DNA recognition mechanism, by a DNA repair polymerase, is the fact that the enzyme must recognize the damaged DNA, containing a small ssDNA gap, in the context of the large excess of the dsDNA. Although the catalytic properties of the domains are established, the role of both domains in the recognition of the DNA substrates is just now emerging (17-20). Quantitative equilibrium studies have shown that both human and rat pol  bind the ssDNA in two binding modes (17, 20). The binding modes differ in the number of occluded nucleotide residues and have been referred to as the (pol )₁₆ and (pol )₅ binding modes (19-23). The existence of the two binding modes is a consequence of the presence of the two structural domains of the protein possessing the DNA-binding subsites, with different DNA binding capabilities (17, 20). Both DNA-binding subsites form the total DNA-binding site of the polymerase. In the (pol )₁₆ binding mode, both the 8- and 31-kDa domains are involved in interactions with the ssDNA, i.e. the total DNA-binding site is engaged in interactions with the nucleic acid. In the (pol )₅ binding mode, only the 8-kDa domain is engaged in interactions with the DNA (17, 20). The subsite located on the 8-kDa domain has similar affinity for both ss and dsDNA, while the subsite on the 31-kDa domain seems to have a preference for the dsDNA, although this has not been rigorously proven.

The mechanism of the formation of the (pol )₁₆ and (pol )₅ binding modes, by human pol , is a complex, multiple-step sequential process (21). In both ssDNA-binding modes the formation of the protein-nucleic acid complex is initiated through the DNA-binding subsite located on the 8-kDa domain (21). Thus the DNA-binding subsite on the 8-kDa domain plays the role of the initiation-binding site of the enzyme to the ssDNA. Analysis of the kinetic data revealed that transitions to subsequent intermediates are also generated through interactions at the 8-kDa domain-DNA interface resulting in the engagement of the 31-kDa domain in interactions with the nucleic acid. The DNA-binding initiation role of the subsite located on the 8-kDa domain is reflected in its energetically homogeneous structure and the capability of accommodating DNA oligomers of different lengths with similar affinity (22).

In the base-excision repair processes, human pol  fills the ssDNA gaps formed in the damaged DNA (1, 5, 9). Thus, the physiological substrates for the enzyme are gapped DNAs that have a stretch of ssDNA embedded between the primer and the dsDNA downstream from the primer. Thermodynamic studies of human pol  binding to the gapped DNA substrates indicate that the ability of the 8-kDa domain DNA-binding site to interact with different nucleic acid conformations is crucial for anchoring the enzyme on the gap (19, 20). In these complexes, the 8-kDa domain binds to the ss and/or dsDNA part of the DNA, downstream from the primer, depending on the length of the ssDNA gap. The 31-kDa domain of the enzyme associates with the dsDNA part of the gapped DNA substrate that contains the primer. Thus, similar to the formation of the (pol )₁₆ binding mode, engagement of the entire total DNA-binding site of the enzyme provides a large increase of the affinity for the specific recognition of the gapped DNA structure.

Understanding the mechanistic aspects of the pol -gapped DNA recognition process is of great importance for elucidation of the polymerase mechanism at the molecular level. Such analysis will also provide important insights into the recognition mechanisms of specific DNA substrates by other nucleic acid-dependent polymerases. The fundamental questions that arise here are the following. How does the mechanism of the gapped DNA recognition differ from the mechanisms of the enzyme binding to the ssDNA in its different binding modes? What is the formation rate of the different intermediates? What are the energetics of the conversions between the different intermediates? How does the length of the ssDNA gap and the presence of the 5'-terminal phosphate group affect the mechanism? Is there a particular step that makes a dominant contribution to the recognition process? Despite its paramount importance, the direct analysis of the dynamics of the gapped DNA substrate recognition by human pol  has not been quantitatively addressed before.

In this article, we report the stopped-flow kinetic analyses of human pol  interactions with the gapped DNA substrates that differ by the number of the nucleotide residues in the ssDNA gap. We provide direct evidence that the mechanism of the specific gap complex formation by human pol  is a three-step, sequential reaction. The bimolecular step includes a very fast association with the DNA, through the 8-kDa domain, followed by two docking steps. The dynamics of the bimolecular step is independent of the length of the ssDNA gap. The internal transition, directly following the bimolecular step, is the major docking step, and is characterized by a large, favorable free energy change for the examined gapped DNA substrates. In this step the DNA-binding subsite located on the 31-kDa domain engages in interactions with the DNA. The 5'-terminal PO group downstream from the primer does not guide the polymerase to the gap, although it stabilizes the first intermediate in the case of the gapped DNA substrate with a longer gap. The data indicate that the phosphate group affects the enzyme orientation in the complex with the DNA and the structure of the ssDNA gap in the complex with the enzyme.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents and Buffers-- All solutions were made with distilled and deionized >18 M (Milli-Q Plus) water. All chemicals were reagent grade. Buffer C is 10 mM sodium cacodylate adjusted to pH 7.0 with HCl, 10% glycerol, 0.1 mM EDTA-Na₂. The temperature and concentrations of NaCl and MgCl₂ in the buffer are indicated in the text.

Human Polymerase -- Human pol  was purified as previously described (17-20). The concentration of the protein was determined using the extinction coefficient, ₂₈₀ = 2.1 × 10⁴ cm¹ M¹, determined by the approach based on the Edelhoch method (23-27).

Nucleic Acids-- The ssDNA oligomers were purchased from Midland Certified Reagents (Midland, TX). Oligomers were at least >95% as judged by autoradiography on polyacrylamide gels. The fluorescein residue is introduced through the fluorescein phosphoramidite, i.e. the label replaces one of the bases in the oligomers. The concentration of the nucleic acids was determined as previously described by us (28, 29).

Fluorescence Measurements-- Steady-state fluorescence titrations were performed using the SLM-AMINCO 8100 spectrofluorometer (26-30). The fractional fluorescence increase of the nucleic acids is defined as F = (F_i  F_o)/F_o, where F_i is the value of the fluorescence at a given titration point and F_o is the initial fluorescence of the sample before addition of the protein (31).

Stopped-flow Kinetics-- Fluorescence stopped-flow kinetic experiments were performed using the SX.MV18 stopped-flow instrument (Applied Photophysics Ltd., Leatherhead, UK). The instrument has a dead-time of ~1.5 ms. The reaction was monitored using the fluorescence emission of the ssDNA oligomers containing the fluorescein residue, with _ex = 485 nm, and the emission observed through a GG495 cutoff filter (Schott, PA), with the excitation monochromator slits at 1.5 mm (band pass ~6.5 nm). A total of 4000 points were collected in each trace that also contains the steady-state value of the sample fluorescence recorded ~2 ms before the flow stops. We usually collect and average 9-15 traces for each sample. The kinetic curves were fitted to extract relaxation times and amplitudes, using the nonlinear, least-square fit software provided by the manufacturer, with the exponential function defined as the following,

(Eq. 1)

where F(t) is the fluorescence intensity at time t, F() is the fluorescence intensity at t = , A_i is the amplitude corresponding to ith relaxation process, _i is the time constant (reciprocal relaxation time) characterizing the ith relaxation process, and n is the number of relaxation processes. All further analyses of the data were performed using Mathematica (Wolfram, Urbana, IL) and KaleidaGraph (Synergy Software, PA) on Macintosh G3 and G4 computers.

Analysis of Kinetic Data-- Analyses of the stopped-flow kinetic data have been performed using the matrix projection operator technique (21, 32-36). This approach is particularly useful in analyzing the amplitudes of the studied reactions and has been extensively described by us (21, 32-36).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Formation of the Gap Complex between Human Pol  and Gapped DNA-- The gapped DNA substrates used to examine the mechanism of the gapped DNA recognition by human pol  are depicted in Fig. 1. The substrates contain two dsDNA parts, each having 10 base pairs (bp). The primary structures of the dsDNA parts are identical in all gapped DNA substrates. The ssDNA gap separates the dsDNA parts. The gap has two, or five residues, i.e. the substrates differ by three residues in the gap. This difference corresponds to the lowest estimate of the site-size of the (pol )₅ binding mode (5 ± 2) that the enzyme forms with the ssDNA (17-20). The bases of the nucleotide residues in the ssDNA gap are all adenosines with the exception of the residue at the 5' end of the gap that is replaced by fluorescein. It provides the signal to monitor the binding and kinetics. Analogous gapped DNA substrates containing the 5'-terminal phosphate group downstream from the primer are included in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   The two gapped DNA substrates that are used to examine the interactions of human pol  with the gapped DNA. The DNA substrates have two dsDNA parts at the 5' end (downstream from the primer) and at the 3' end (primer location) of the template strand, which are identical in both substrates. The dsDNA parts are separated by the ssDNA of the gap containing five (Substrate A), and two nucleotide residues (Substrate B). Substrates C and D are analogous to Substrates A and B, but contain the 5'-terminal phosphate group downstream from the primer.

Our previous studies have shown that binding of the enzyme to the etheno derivatives of the considered gapped DNAs is accompanied by a nucleic acid fluorescence increase (19-21). However, the observed signal is not adequate enough to quantitatively examine the complex kinetics of the reaction. On the other hand, we have found that binding of the enzyme to the gapped DNA, containing the fluorescein residue in place of one of the nucleotide residues in the ssDNA gap, as shown in Fig. 1, is accompanied by a large fluorescence increase of the DNA. This signal change provides the required resolution to monitor the kinetics of the enzyme-gapped DNA complex formation. In this context, fluorescein derivatives of the DNA, where the label is substituted for one of the bases, could be very useful in examining other protein-nucleic acid interactions. We have used analogous ssDNA oligomers containing the fluorescein residue in our analyses of the kinetics of the polymerase binding to the ssDNA (21).

At maximum saturation, the considered gapped DNA substrates, depicted in Fig. 1, can bind up to three molecules of human pol  (20). However, the binding process is very complex. A single enzyme molecule forms a high affinity complex that includes the ssDNA gap, while the remaining two polymerase molecules are bound with a significantly lower affinity to two dsDNA parts. We refer to the high affinity complex as the gap complex (20, 21). The high affinity of the gap complex results from the fact that only in this complex the enzyme engages both the 8- and 31-kDa domains in interactions with the nucleic acid. The 8-kDa domain interacts with the dsDNA part of the 5' end of the template strand, downstream from the primer, while the 31-kDa domain engages in interactions with the dsDNA part at the 3' end of the template strand which includes the primer (Fig. 1). The large difference between the affinities of the two binding processes strongly separates them with respect to the protein concentration (20). This strong separation of the two binding processes allows us to study both the thermodynamics and kinetics of the specific human pol  binding directly to the gap and the formation of the gap complex, independently from the nonspecific enzyme binding to the dsDNA parts.

Fluorescence titration of the gapped DNA substrate, containing five residues in the ssDNA gap (Fig. 1, substrate A), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl₂, is shown in Fig. 2a. The binding curve has been analyzed using a single-binding site isotherm,

(Eq. 2)

where F_max is the maximum relative fluorescence increase and K_G5 is the overall equilibrium binding constant characterizing the enzyme affinity for the gapped DNA substrate, i.e. the gap complex with five residues in the ssDNA. The solid line is the computer fit of the experimental isotherm to Equation 2, with K_G5 = (1 ± 0.2) × 10⁹ M¹ and F_max = 0.63 ± 0.05. It is clear that the theoretical line provides an excellent description of the experimental isotherm indicating that the formation of the gap complex is exclusively observed (20). In other words, in the examined protein concentration range, binding of the additional enzyme molecules to the dsDNA parts of the DNA substrate, with the affinities characterized by binding constants in the range of 10⁵-10⁶ M¹, does not occur (21).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   a, fluorescence titration of a gapped DNA substrate with five-nucleotide residues in the ssDNA gap () (Fig. 1, Substrate A), and the same substrate containing the 5'-terminal phosphate group () (Fig. 1, Substrate C), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2. The concentrations of the DNA substrates are 1.11 × 108 M. The solid lines are the computer fit of the experimental data to the single binding-site isotherm with Fmax = 0.63 and KG5 = 1 × 109 M1 () and Fmax = 1.76 and KG5 = 1 × 109 M1 (), respectively. Titrations of the same fluorescent gapped DNA substrates (1.11 × 108 M) with human pol  in the presence of the competing, analogous, and unmodified DNA substrates, containing the 5'-terminal phosphate group (), without the phosphate group () (see text for details). The concentrations of the unmodified DNAs are 1.11 × 108 M () and 2.1 × 108 M (), respectively. The solid lines are the computer fits of the experimental data using KG5 = 1 × 109 M1 for both unmodified DNAs (30, 31). b, fluorescence titration of a gapped DNA substrate with two nucleotide residues in the ssDNA gap () (Fig. 1, Substrate B), and the same substrate containing the 5'-terminal phosphate group () (Fig. 1, Substrate D), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2. The solid lines are the computer fits of the experimental data to the single binding-site isotherm (Equation 2) with Fmax = 0.73 and KG2 = 3 × 109 M1 (), and Fmax = 1.5 and KG2 = 3 × 109 M1 (), respectively.

A 5'-terminal phosphate group, downstream from the primer in the damaged DNA is a common intermediate product in base excision repair (1, 9, 12). Fluorescence titration of the gapped DNA substrate containing five residues in the ssDNA gap and also the 5'-terminal phosphate group (Fig. 1, Substrate C), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl₂, is included in Fig. 2a. The binding curve has been analyzed using a single-binding site isotherm, defined by Equation 2, that provides K_G5 = (1 ± 0.2) × 10⁹ M¹ and F_max = 1.76 ± 0.05. Thus, the presence of the 5'-terminal PO group has no effect on the overall affinity (20). However, the PO group has a dramatic effect on the fluorescence change accompanying the formation of the complex with F_max being by a factor of ~3 higher than that observed in the absence of the phosphate group (Fig. 2). Such a large difference in the spectroscopic properties of the polymerase-DNA complex, in the absence and presence of the PO group, indicates that the conformations of the nucleic acid, particularly the structures of the ssDNA gap in both complexes, are significantly different. This is despite the fact that the affinities are virtually identical.

Because the fluorescein residue may affect the affinities of human pol  for the examined gapped DNA substrates, we performed fluorescent titrations in the presence of the competing non-fluorescent gapped DNA substrate with five residues in the ssDNA gap (Fig. 1), but containing adenosine in place of the fluorescein residue. The corresponding competition titration curves, for the gapped DNA without and with 5'-terminal phosphate group, are included in Fig. 2a. The solid lines are computer fits of the experimental isotherms which provide binding constant, K_G5 = (1 ± 0.2) × 10⁹ M¹, for both unmodified DNAs (30, 31). Thus, the presence of the fluorescein residue does not affect, to any detectable extent, the affinity of the protein for the gapped DNA substrate independently of the presence of the phosphate group (see below).

Fluorescence titration of the gapped DNA substrate, containing two residues in the ssDNA gap (Fig. 1, Substrate B), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl₂, is shown in Fig. 2b. The solid line is the computer fit of the experimental isotherm using the equation analogous to Equation 2, with K_G2 = (3 ± 0.3) × 10⁹ M¹ and F_max = 0.73 ± 0.05. Thus, the overall binding constant is by a factor of ~3 higher for the gapped DNA substrate with a smaller gap (21). Also, the value of the maximum relative fluorescence increase, F_max = 0.73 ± 0.05, is higher than the value of 0.63 ± 0.05 obtained for the DNA substrate with five residues in the gap, indicating differences in the structure of the ssDNA gap between two DNA substrates in the complex with the polymerase (Fig. 2a).

Fluorescence titration of the gapped DNA substrate, containing two residues in the ssDNA gap and the 5'-terminal phosphate group, downstream from the primer (Fig. 1, Substrate D), with human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl₂, is included in Fig. 2b. The solid line is the computer fit of the experimental isotherm (Equation 2), with K_G2 = (3 ± 0.3) × 10⁹ M¹ and F_max = 1.5 ± 0.07. Similar to the DNA substrate with five residues in the gap the presence of the phosphate group does not, to a detectable extent, affect the overall affinity of the enzyme for the substrate with the smaller gap. However, the maximum relative fluorescence, F_max, increase is by a factor of ~2 higher than the corresponding parameter obtained in the absence of the phosphate group. Thus, similar to the DNA substrate with a long ssDNA gap, the presence of the 5'-terminal PO group strongly affects the conformation of the protein-nucleic acid complex, although to a smaller extent. Also, competition titrations of the unmodified DNA substrates, containing two adenosines in the ssDNA gap, show that the fluorescein residue in the gap does not affect, to any extent, the affinity of the enzyme for the DNA (data not shown).

Kinetics of Human Pol  Binding to the Gapped DNA Substrate with Five Residues in the ssDNA Gap-- The fluorescence stopped-flow kinetic trace of the gapped DNA substrate, having the ssDNA gap with five residues, after mixing 1.5 × 10⁸ M nucleic acid with 1.6 × 10⁷ M human pol  (final concentrations) in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl₂, is shown in Fig. 3a. The plot is shown in logarithmic scale with respect to time. The initial horizontal part of the trace corresponds to the steady-state fluorescence intensity, recorded for ~2 ms, before the flow stops (32-36). It is evident that the observed kinetics are complex and clearly show the presence of multiple steps. The solid line in Fig. 3a is a nonlinear, least-square fit of the experimental curve using a two-exponential fit as defined by Equation 1. The single-exponential function does not provide an adequate description of the observed kinetics, as indicated by the included single-exponential fit (dashed line).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   a, the fluorescence stopped-flow kinetic trace, after mixing human pol  with the gapped DNA substrate having five nucleotide residues in the ssDNA gap (Fig. 1, Substrate A), in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl (ex = 485 nm, em > 495 nm). The final concentrations of the polymerase and the DNA are 1.6 × 107 M and 1.5 × 108 M (Gapped DNA), respectively. The solid line is the two-exponential nonlinear, least-square fit of the experimental curve, using Equation 1. The dashed line is the nonlinear, least-square fit using the single-exponential function. The horizontal, initial part of the trace is the steady-state value of the fluorescence of the sample recorded ~2 ms before the flow stopped. b, the same fluorescence stopped-flow trace as in a, together with the zero line trace (lower trace), which is obtained after mixing the nucleic acid, at the same concentration as used with the protein, but only with the buffer. The solid line is the same three-exponential nonlinear, least-square fit of the experimental curve as shown in a.

The stopped-flow kinetic curve, together with the trace corresponding to the DNA substrate alone, at the same concentration of the nucleic acid as used with the protein, but only mixed with the buffer, is shown in Fig. 3b. Recall, the total amplitude of the reaction, A_Tot, is the difference between the fluorescence intensity recorded at the end point of the kinetic trace and the zero line recorded for the nucleic acid alone (32-36). Therefore, although the two-exponential fit provides an excellent description of the observed kinetic process, it yields the sum of the amplitudes that is larger than the observed total amplitude of the overall, relaxation process. This behavior is observed at all studied enzyme concentrations (data not shown). In other words, these data indicate that the observed reaction contains at least one additional step, characterized by the relaxation time, ₁, that precedes the observed steps. The values of ₁ are too short to be extracted in the stopped-flow experiment. Notice that this fast step must be characterized by negative amplitude (see below). Therefore, the formation of the gap complex between human pol  and the considered gapped DNA substrate is a process that includes at least three relaxation steps.

The dependence of the reciprocal relaxation times, 1/₂ and 1/₃ upon the total concentration of human pol , is shown in Fig. 4, a and b. The functional dependence of 1/₂ upon [human pol ] is seemingly linear in the examined enzyme concentration range. We could not extend the measurements to higher enzyme concentrations because binding of the enzyme to the dsDNA part of the gapped DNA substrate would begin to interfere with the observed kinetics (20). However, as we discussed above, the relaxation process characterized by the relaxation time, ₂, is preceded by a faster process, characterized by ₁. Therefore, the presence of the initial, very fast process indicates that ₂ characterizes an intramolecular transition. The seemingly linear dependence of ₁ upon the [enzyme] indicates that in the limited range of the enzyme concentration examined only the initial part of the functional dependence of 1/₂ upon [enzyme] is recorded (21, 32-35). On the other hand, the independence of 1/₃ of the polymerase concentration clearly indicates that this relaxation time characterizes another intramolecular transition of the complex (21, 32-35). Therefore, the simplest mechanism which can describe the observed dependence of the relaxation times upon the pol  concentration and the presence of the third fast step is a three-step, sequential reaction in which bimolecular binding is followed by two first-order transitions described by the Scheme I. 

Although the relaxation time for the first, fast normal mode cannot be extracted from the data, the amplitude of the first mode, A₁, can be obtained as a difference between the total amplitude of the reaction, A_Tot, and the known amplitudes of the second and third normal modes, A₁ and A₂ (32-36) as the following equation.

(Eq. 3)

Fig. 4c shows the dependence of the normalized, individual amplitudes, A₁, A₂, and A₃, of the three relaxation steps upon the human pol  concentration. The amplitude of the first relaxation step, A₁, is negative. Also, its absolute values slightly increase with increasing concentrations of human pol . The positive amplitude, A₂, dominates the relaxation process over the entire examined range of [human pol ]. The values of A₂ slightly increase with the increasing of the [enzyme] and at high [human pol ] are larger than 1, i.e. the amplitude is larger than the total amplitude A_Tot. This results from the fact that the values of A₂ compensate for the negative values of A₁ (21). The values of A₃ are also positive and have little dependence on the enzyme concentration. The observed behavior of the individual amplitudes as functions of the human pol  concentration is in agreement with the proposed mechanism defined by Scheme I (21).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   The dependence of the reciprocal relaxation times for the binding of the gapped DNA substrate having five nucleotide residues in the ssDNA gap (Fig. 1, Substrate A) to human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2, upon the total concentration of the enzyme. The solid lines are nonlinear, least-square fits according to the three-step sequential mechanism, defined by Scheme I, with the rate constants: k1 = 6 × 109 M1 s1, k1 = 1000 s1, k2 = 1150 s1, k2 = 15 s1, k3 = 0.23 s1, and k3 = 0.17 s1 (details in text). a, 1/2. b, 1/3. The error bars are standard deviations obtained from three to four independent experiments. c, the dependence of the normalized, individual relaxation amplitudes, A1, A2, and A3, for the binding of the gapped DNA substrate having five nucleotide residues in the ssDNA gap (Fig. 1, Substrate A) to human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2, upon the logarithm of the total enzyme concentration. The solid lines are nonlinear, least-square fits according to the three-step sequential mechanism, defined by Scheme I, with the relative fluorescence intensities F1 = 1, F2 = 1, F3 = 1.55, and F4 = 1.64. The maximum fluorescence increase of the nucleic acid is taken from the equilibrium fluorescence titration in the same solution conditions as Fmax = 0.63 (Fig. 2a). The rate constants are the same as in a and b. A1, ; A2, ; A3, .

To extract all rate and spectroscopic parameters characterizing the partial steps and the intermediates of the kinetic system, we applied the following strategy utilizing the fact that both the amplitudes and the relaxation times contain information about the examined system (32-35). The analysis is initiated by numerical nonlinear, least-square fitting of the individual relaxation times. Because the first step is very fast, the fits were performed with the starting value of the rate constant, k₁, near the diffusion-controlled limit, e.g. ~1 × 10¹⁰ M¹. We also know the value of the macroscopic binding constant, K_G5 = (1 ± 0.2) × 10⁹ M¹, for the enzyme binding to the gap, independently obtained in the same solution conditions by the equilibrium fluorescence titration method (Fig. 2a). The macroscopic binding constant is related to the partial equilibrium steps by,

(Eq. 4)

where K₁ = k₁/k₁, K₂ = k₂/k₂, and K₃ = k₃/k₃. The above relationship reduces to five the number of independent parameters in fitting the relaxation times. Subsequently, the obtained rate constants were used as starting values in the fitting of the three individual amplitudes and extract relative molar fluorescence parameters, i.e. to assess the conformational state of the protein-nucleic acid complex in each intermediate. This was accomplished using the matrix projection operator technique (32-36). This part of the analysis uses the value of the maximum fractional increase of the nucleic acid fluorescence accompanying the complex formation, F_max = 0.6 ± 0.03, that is known from independent equilibrium titrations (Fig. 2a). The F_max parameter can be analytically expressed (32-35) as,

(Eq. 5)

where F₂ = (F₂  F₁)/F₁, F₃ = (F₃  F₁)/F₁, and F₄ = (F₄  F₁)/F₁ are fractional fluorescence intensities of each intermediate in the formation of the gap complex, relative to the molar fluorescence intensity of the free DNA substrate, F₁. It should be pointed out that, contrary to the F_i values, the fluorescence parameters, F₂, F₃, and F₄, are relative molar fluorescence intensities, but not fractional intensities, with respect to the free nucleic acid fluorescence. Equation 5 provides an additional relationship among the fluorescence parameters, thus, decreasing the number of independent variables. The refinement of the values of rate constants and molar fluorescence parameters was accomplished by global fitting that simultaneously includes all relaxation times and amplitudes. The solid lines in Fig. 4, a-c, are computer fits of the relaxation times and amplitudes, according to the above mechanism, using a single set of rate and spectroscopic parameters. The obtained rate constants and relative molar fluorescence intensities for the mechanism defined by Scheme I are included in Table I.

View this table: [in this window] [in a new window]   Table I Kinetic, thermodynamic, and spectroscopic parameters characterizing the binding of human pol  to gapped DNA substrates (Fig. 1) differing by the number of nucleotide residues in the ssDNA gap (gap-size), in the absence and presence of the 5'-terminal phosphate group, in buffer C (pH 7, 10 °C), containing 100 mM NaCl and 1 mM MgCl2 Determined in independent equilibrium fluorescence titrations (details in text).

The forward rate constant, k₁ = 6 × 10⁹ M¹ s¹, characterizing the bimolecular binding step is very high, close to the value predicted for the diffusion-controlled reaction (37, 38) (see "Discussion"). The value of the rate constant, k₁ = 1000 ± 100 s¹, indicates that the enzyme can easily be released back to the solvent from the first intermediate (G)₁. The transition to the second intermediate, (G)₂, is also a fast process with the forward rate constant, k₂ = 1150 ± 120 s¹ (Table I). However, the transition to (G)₃ is dramatically slower with the forward rate constant, k₃, being ~4 orders of magnitude lower than k₂. The obtained rate constants for each step provide partial equilibrium constants K₁ = (6 ± 1.4) × 10⁶ M¹, K₂ = 77 ± 26, and K₃ = 1.4 ± 0.6 (Table I). Thus, the first step has a predominant contribution to the free energy of the enzyme binding to the examined gapped DNA substrate, G° (Fig. 1, Substrate A). Notice that the second step also provides a significant contribution to the G°. The (G)₂  (G)₃ transition is much less energetically favorable (see "Discussion").

Amplitude analysis indicates that the formation of the first intermediate (G)₁ is not accompanied by any molar fluorescence change of the DNA substrate (F₂ = 1 ± 0.03), as compared with the free nucleic acid (Table I). However, the formation of the subsequent (G)₂ intermediate is accompanied by a molar fluorescence increase (F₃ = 1.55 ± 0.05), indicating different structures of the protein-nucleic acid complex in both intermediates (see "Discussion"). Conformational transition to (G)₃ induces a very low, additional molar fluorescence increase of the nucleic acid over F₃ (F₄ = 1.64 ± 0.05), indicating that the structure of the examined protein-gapped DNA complex is similar in both (G)₂ and (G)₃ intermediates.

Kinetics of Human pol  Binding to the Gapped DNA Substrate with Two Residues in the ssDNA Gap-- Analogous stopped-flow kinetic studies have been performed with gapped DNA substrate with two residues in the ssDNA gap (Fig. 1, Substrate B). The experiments have been performed in the same solution conditions, i.e. buffer C (pH 7.0, 10 °C) containing 100 mM NaCl and 1 mM MgCl₂. The experimental kinetic traces required a two-exponential fit (data not shown). However, the two-exponential fit yields the sum of amplitudes significantly larger than the total amplitude resulting from the complex formation at all examined enzyme concentrations. Thus, as we discussed above, the data indicate that there is at least one additional very fast step preceding the observed trace, characterized by the relaxation time, ₁, and negative amplitude.

The reciprocal relaxation times, 1/₂ and 1/₃, for the association of human pol  with the gapped DNA substrate, with two residues in the ssDNA gap (Fig. 1, Substrate B), as functions of the total human pol  concentration, are shown in Fig. 5, a and b. The dependence of the amplitudes upon [human pol ] is shown in Fig. 5c. The values of 1/₂ show a more pronounced hyperbolic dependence upon [human pol ] than observed in the case of the DNA gapped substrate with a longer gap (Fig. 4a) which results from the higher affinity of the enzyme for the DNA with a shorter gap. This behavior and the existence of the preceding fast process indicate that the relaxation time, ₂, describes an intramolecular isomerization. The values of 1/₃ are independent of the [enzyme], an indication that this relaxation time characterizes another intramolecular transition (32-35, 38). Therefore, association of human pol  with the gapped DNA having two residues in the gap, is described by the same mechanism as depicted by Scheme I. 

View larger version (16K): [in this window] [in a new window]   Fig. 5.   The dependence of the reciprocal relaxation times for the binding of the gapped DNA substrate having two nucleotide residues in the ssDNA gap (Fig. 1, Substrate B) to human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2, upon the total concentration of the enzyme. The solid lines are nonlinear, least-square fits according to the three-step sequential mechanism, defined by Scheme I, with the rate constants: k1 = 6 × 109 M1 s1, k1 = 1000 s1, k2 = 600 s1, k2 = 5 s1, k3 = 3.4 s1, and k3 = 1.1 s1 (details in text). a, 1/2. b, 1/3. The error bars are standard deviations obtained from three to four independent experiments. c, the dependence of the normalized, individual relaxation amplitudes, A1, A2, and A3, for the binding of the gapped DNA substrate having two nucleotide residues in the ssDNA gap (Fig. 1, Substrate B) to human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2, upon the logarithm of the total enzyme concentration. The solid lines are nonlinear, least-square fits according to the three-step sequential mechanism, defined by Scheme I, with the relative fluorescence intensities F1 = 1, F2 = 0.6, F3 = 1.66, and F4 = 1.74. The maximum fluorescence increase of the nucleic acid is taken from the equilibrium fluorescence titration in the same solution conditions as Fmax = 0.73 (Fig. 2b). The rate constants are the same as in a and b. A1, ; A2, ; A3, .

The analysis of the experimental stopped-flow data has been performed as described above. The obtained kinetic and spectroscopic parameters, obtained from the examination of the data shown in Fig. 5, are included in Table I. The rate constant, k₁ and k₁, of the bimolecular step are very similar to the rate constants determined for the association of the enzyme with the gapped DNA substrate with five residues in the gap (Table I). Thus, the dynamics of the initial binding step are not affected by the large difference in the size of the ssDNA gap, although the structure of the (G)₁ intermediate is different from the analogous intermediate in the enzyme association with the DNA substrate with five residues in the ssDNA gap, as indicated by the significantly lower value of F₂. On the other hand, the value of the forward rate constant, k₂ = 600 s¹, is lower as compared with the value of k₂ = 1150 s¹ observed for the DNA substrate with five residues in the ssDNA gap. Thus, the lower size of the gap hinders the transition to the (G)₂ intermediate in the human pol -gapped DNA complex. However, the value of k₂ is also lower by a factor of ~3, as compared with the DNA substrate with the longer gap. As a result, the partial equilibrium constant, K₂, assumes a higher value, indicating that the (G)₂ intermediate is energetically more favorable for the DNA substrate with only two residues in the gap. Nevertheless, the very similar values of the relative molar fluorescence intensities of the (G)₂ intermediate for both gapped DNA substrates indicate that the structure of this intermediate is similar, i.e. independent of the length of the ssDNA gap (Table I). A pronounced effect of the size of the ssDNA gap is observed on the transition to the third intermediate, (G)₃. Both k₃ and k₃ are larger by a factor of ~20, i.e. the process becomes much faster in both directions with a larger increase toward the formation of (G)₃, resulting in a larger value of K₃ for the gapped DNA substrate with a shorter gap. The (G)₃ intermediate for this DNA substrate becomes a significant part of the population of the enzyme-DNA complex at equilibrium.

Effect of the 5'-Terminal Phosphate Group Downstream from the Primer on the Kinetics of Human pol  Binding to Gapped DNA Substrates-- The presence of the 5'-terminal phosphate group, downstream from the primer, plays an important role in DNA substrate recognition and catalysis (11, 14). However, direct thermodynamic analysis of the effect of the 5'-terminal phosphate on the binding of human pol  to DNA substrates shows only a moderate effect on the overall binding constant of the enzyme for the gap (Fig. 2b) (20). The role of the 5'-terminal phosphate group in the dynamics of the gapped DNA recognition process by human pol  has been examined using the gapped DNA substrates shown in Fig. 1, but with the 5'-terminal phosphate group on the oligomer downstream from the primer (Fig. 1, Substrates C and D).

The experimental stopped-flow kinetic traces require a two-exponential fit for both DNA substrates, with five and two residues in the ssDNA gap (data not shown). Moreover, the sum of the observed amplitudes is significantly larger than the total amplitude resulting from the complex formation for all examined enzyme concentrations. Thus, as we discussed above, the data indicate that there is at least one additional very fast step preceding the observed trace, characterized by the relaxation time, ₁, that must be characterized by negative amplitude. The reciprocal relaxation times, 1/₂ and 1/₃, for the association of human pol  with the gapped DNA substrate with five residues in the ssDNA gap and the 5'-terminal phosphate group downstream from the primer (Fig. 1, Substrate C) as functions of the total human pol  concentration, are shown in Fig. 6, a and b. The dependence of the observed amplitudes upon the [human pol ] is shown in Fig. 6c. In the examined [human pol ] range the values of 1/₂ show typical, hyperbolic dependence upon [human pol ] and clearly indicate that the relaxation time, ₂, describes an intramolecular isomerization. Similar to the gapped DNAs discussed above, independence of 1/₃ upon the enzyme concentration clearly indicates that this relaxation time characterizes another intramolecular transition (21). Therefore, association of human pol  with the considered gapped DNA, having the 5'-terminal phosphate downstream from the primer, is described by the mechanism defined by Scheme I. Analogous behavior of the relaxation times and amplitude has been observed for the gapped DNA substrate with only two residues in the ssDNA gap (data not shown). The obtained kinetic and spectroscopic parameters for both DNA substrates are included in Table I.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The dependence of the reciprocal relaxation times for the binding of the gapped DNA substrate having five nucleotide residues in the ssDNA gap with the 5'-terminal phosphate group (Fig. 5, Substrate C) to human pol  in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl and 1 mM MgCl2, upon the total concentration of the enzyme. The solid lines are nonlinear least-square fits according to the four-step sequential mechanism defined by Scheme I with the rate constants: k1 = 6 × 109 M1 s1, k1 = 300 s1, k2 = 310 s1, k2 = 10 s1, k3 = 5 s1, and k3 = 5 s1. a, 1/2. b, 1/3. The error bars are standard deviations obtained from three to four independent experiments. c, the dependence of the normalized, individual relaxation amplitudes, A1, A2, and A3, for the binding of human pol  to the gapped DNA substrate having five nucleotide residues in the ssDNA gap with the 5'-terminal phosphate group (Fig. 5, Substrate C), in buffer C (pH 7.0, 10 °C), containing 100 mM NaCl, upon the logarithm of the total concentration of the enzyme. The solid lines are nonlinear, least-square fits according to the four-step sequential mechanism, defined by Scheme I, with the relative molar fluorescence intensities F1 = 1, F2 = 1.1, F3 = 2.45, and F4 = 3.13. The maximum fluorescence increase of the nucleic acid is taken from the equilibrium fluorescence titration in the same solution conditions as Fmax = 1.76 (Fig. 2a). The rate constants are the same as in a and b. A1, ; A2, ; A3, .

The obtained data indicate that the forward rate constant, k₁, of the bimolecular step is not affected by the presence of the phosphate group, independently of the size of the ssDNA gap. However, the value of k₁ is lower by a factor of ~3 for the DNA substrate with five residues in the ssDNA gap, i.e. the presence of the phosphate group increases the stability of the first intermediate, (G)₁, as compared with the corresponding DNA substrate without the 5'-terminal phosphate group (Table I). The presence of the phosphate group has a profoundly different effect on the formation of the second intermediate, (G)₂, for both gapped DNAs. In the case of the DNA molecule with five residues in the ssDNA gap, the forward rate constant, k₂, is lower by a factor of ~4. Thus, the presence of the phosphate group hinders the transition to the (G)₂ intermediate for a longer gap. The phosphate group also decreases the value of k₂. As a result, the value of the partial equilibrium constant, K₂, is decreased by a factor of ~2. Thus, both the dynamics of the (G)₁  (G)₂ transition and the free energy change accompanying the formation of the (G)₂ intermediate are affected by the PO group. In the case of the gapped DNA substrate with two residues in the ssDNA gap, both rate constants, k₂ and k₂, are not changed by the presence of the phosphate group (Table I). Within experimental accuracy, the value of the partial equilibrium constant, K₂, is the same as observed in the absence and presence of the PO group. Similarly, little effect of the phosphate group is observed for the (G)₂  (G)₃ transition. Thus, the presence of the PO group has no effect on either the dynamics or the free energy changes accompanying the formation of the (G)₂ and (G)₃ intermediates when the gap is only two residues long.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Mechanism of the Gap Complex Formation by Human pol  Is a Multiple Step Kinetic Process-- Elucidation of the dynamics of human pol  interactions with gapped DNA substrates is of paramount importance for understanding the gapped DNA recognition process by the enzyme at the molecular level. The kinetic data obtained in this work indicate that the mechanism of the gap complex formation with the DNA substrates with two and five residues in the ssDNA gap is a minimum three-step, sequential process in examined solution conditions described by Scheme I. Thus, the kinetic mechanism of the gap complex formation is not affected by the large difference between the sizes of the ssDNA gap. The independence of the mechanism of the gap recognition of the size of the gap provides the first indication that the size of the ssDNA gap does not constitute any specific recognition element for the enzyme. However, the bimolecular step is followed by at least two conformational transitions of the enzyme-ssDNA complex that differ dramatically in the values of the rate constants and accompanying free energy changes. Analysis of the entire mechanism provides the clue for understanding the specific formation of the gap complex.

The bimolecular step of the gap complex formation by human pol  is very fast and close to the diffusion-controlled reaction (32-41). The determined forward rate constant, k₁ ~ 6 × 10⁹ M¹ s¹, is high and independent of the length of the ssDNA gap. The independence of k₁ upon the length of the gap is fully understandable in the context of our recent studies of the kinetics of human pol  binding to the ssDNA in its (pol )₅ and (pol )₁₆ binding modes (21). These data indicate that, in the bimolecular step, the enzyme exclusively associates with the ssDNA, using its 8-kDa domain. Thus, the first initial contact between the enzyme and the nucleic acid occurs through the DNA-binding subsite of the 8-kDa domain. Moreover, thermodynamic studies of the 8-kDa domain interactions with the ssDNA for the analogous rat pol  showed that the domain has very similar affinities for both ss- and dsDNA (22). The DNA-binding subsite of the domain can accept ss and ds oligomers of a different length with similar affinity. Notice, the site size of the 8-kDa domain with the ssDNA is ~10 nucleotide residues and is ~10 bp with the dsDNA (22). Therefore, in any complex with the examined gapped DNA substrates (Fig. 1), the domain predominantly engages a similar nucleic acid structure, i.e. a mixture of ss and dsDNA conformations. Thus, the initial contact with any gapped DNA substrate occurs through the same DNA-binding subsite of the enzyme that does not differentiate between different DNA conformations resulting in very similar values of k₁, as observed in the case of the ssDNA (21). The similarity of the (G)₁ intermediates is also indicated by the similar values of the dissociation rate constant, k₁, which indicates the same lifetime of the (G)₁ intermediate for both gapped DNAs.

The similarity between the first step in the formation of the (pol )₅ and (pol )₁₆ binding modes, where the 8-kDa domain of the polymerase makes the first contact with the nucleic acid, and the first step in the binding of the enzyme to the gapped DNA substrate is also evident in the kinetics of the isolated 8- and 31-kDa domains binding to the DNA.² Analogous kinetic studies indicate that the first step in the binding of the isolated 8-kDa domain to the ss and dsDNA is characterized by rate constants, k₁ ~ 5 × 10⁹ M¹ s¹ and k₁ ~ 1000 s¹, which are very close to the same parameters observed for the intact enzyme binding to the gapped DNA (Table I). The affinity of the 31-kDa domain for the DNA is much lower than that of the 8-kDa domain. The shortest relaxation time appears only at the protein concentration range that is more than an order of magnitude higher than that observed for the 8-kDa domain. This behavior strongly suggests that the first step in the 31-kDa domain is much slower than observed for the 8-kDa domain, and the intact enzyme.