Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

doi:10.1016/j.jmb.2007.06.054

J. Mol. Biol. (2007) 373, 75–95

Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex Maria J. Jezewska, Paul J. Bujalowski and Wlodzimierz Bujalowski⁎ Department of Biochemistry and Molecular Biology, Department of Obstetrics and Gynecology, The Sealy Center for Structural Biology, Sealy Center for Cancer Cell Biology, The University of Texas Medical Branch at Galveston, 301 University Boulevard, Galveston, TX 77555-1053, USA

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) basepairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme–ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNAbinding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the noncatalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein–protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed. © 2007 Published by Elsevier Ltd.

*Corresponding author

Keywords: DNA polymerases; DNA replication; DNA repair; protein-DNA interactions; quantitative fluorescence titrations

Introduction The African swine fever virus (ASFV) is the etiological agent responsible for a highly lethal disease of domestic pigs.1–7 One of the enzymes encoded by the DNA genome of the virus is a DNA

Abbreviations used: ASFV, African swine fever virus; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; BER, base excision repair; CP, 7-diethylamino-3-(4′-maleimidylphenyl)-4methylcoumarin; CPM, 7-methoxy-coumarin; MCT, macromolecular competition titration method. E-mail address of the corresponding author: [email protected] 0022-2836/$ - see front matter © 2007 Published by Elsevier Ltd.

polymerase, a member of the pol X family referred to as ASFV pol X.5–7 ASFV pol X shows functional similarities to the β-type DNA repair polymerases that include template-directed DNA synthesis, distributive DNA synthesis on template-primer DNA substrates, processivity on the gapped DNAs with the single-stranded DNA (ssDNA) gap, and efficient filling of the single nucleotide gaps.5–13 Such a spectrum of functional activities indicates that ASFV pol X is a part of the DNA repair apparatus of the viral DNA geared to repair viral DNA that has been damaged by the host reaction to infection.5–7 The enzymes of the pol X family show a typical polymerase fold, called a thumb, palm, and fingers, due to its resemblance to the human hand.8,14 However, NMR studies indicate that ASFV pol X consists only of the palm domain, which includes the first 105

76 amino acid residues from the N terminus of the protein and the C-terminal domain, as depicted in Figure 1.15,16 The palm domain contains a triad of invariant aspartate residues involved in the catalysis of the DNA synthesis.15,16 Both the N terminus and the C terminus domains possess unique, positively charged helices, αC and αE, respectively, which are positioned at the surface of the domains and are implicated in DNA binding, although the structure of the co-complex has not been determined. A fundamental problem of DNA repair polymerase–DNA interactions is how the enzyme specifically recognizes the damaged DNA, containing a small ssDNA gap, in the context of the large excess of the double-stranded DNA (dsDNA).17–25 Such

ASFV Pol X-dsDNA Interactions

specificity indicates an intricate recognition process, already found for the mammalian pol β. 18–25 However, while pol β has a typical polymerase fold and an additional 8 kDa domain, crucial for enzyme interactions with the nucleic acid, the structure of the ASFV pol X is dramatically different (Figure 1). Our recent quantitative studies shed light on the complex interactions of the ASFV X with the nucleic acid.17 The total site-size of the ASFV pol X–ssDNA complex is 16(±2) nucleotides, which is surprisingly large for such a small protein. Moreover, the total ssDNA-binding site has a complex heterogeneous structure. It contains the proper ssDNA-binding subsite, a structurally and functionally separated area, characterized by the high nuc-

Figure 1. The NMR three-dimensional structure of ASFV pol X.15 The lysine residues 59, 60, and 63 in the αC helix of the catalytic N-terminal domain are marked in blue; lysine residues 131, 132, and 133, contained in the αE helix of the noncatalytic C-terminal domain, are marked in magenta. The conservative aspartate residues D49, D50, and D101 are marked in black and show the location of the active site of the enzyme. The single tryptophan residue, W92, is marked in red.

ASFV Pol X-dsDNA Interactions

leic acid affinity encompassing 7(±1) nucleotides. On the other hand, the enzyme can engage an additional binding area (subsite) in interactions with the DNA at a distance of about seven or eight nucleotides from the proper site. This functional heterogeneity corroborates well with the NMR structure of the enzyme, which indeed indicates the presence of two positively charged regions in the protein structure (Figure 1). Nevertheless, the ASFV pol X does not form different binding modes with the ssDNA where the enzyme engages only one of its DNA-binding subsites in interactions with the DNA. Such different binding modes were observed for the mammalian pol β and result from the significant autonomy of the DNA-binding subsites of this enzyme.18–25 Interactions of a DNA polymerase with the DNA play a vital role in the functioning of the enzyme, since the polymerase complex with the DNA constitutes the binding and recognition site for dNTPs.26–28 Moreover, in the case of DNA repair polymerase, elucidation of the enzyme interactions with the single and double-stranded conformations of the nucleic acid is of paramount importance for understanding the recognition mechanism of the damaged DNA, because both conformational states of the nucleic acid are likely to be involved in the recognition process.14,17,18–25,29–31 In spite of its significance for understanding the DNA recognition process by ASFV pol X, the direct and quantitative analyses of the enzyme interactions with the dsDNA have not been addressed. The fundamental aspects of these interactions, like stoichiometries, intrinsic affinities, and cooperativities, the role of the DNAbinding subsites in the recognition process are unknown, hindering any rational mechanistic analyses of the enzyme activities.

Results The site-size of the ASFV Pol X–dsDNA complex Interactions of ASFV pol X with the dsDNA are not accompanied by adequate changes of the protein fluorescence that would allow us to examine the complex binding process. However, we have found that association of the ASFV pol X with dsDNA oligomers, labeled at the 5′ end of one of the ssDNA strands with the coumarin derivative 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CP) is accompanied by significant quenching of the nucleic acid fluorescence.29–31 The induced emission changes provide an adequate signal to perform high-resolution measurements of the enzyme– dsDNA complex formation. The selected dsDNA oligomers are shown in Figure 2. These oligomers contain a random sequence of bases and constant, ∼70%, content of the G-C base-pairs. The length of the shortest and longest oligomers are 10 bp and 20 bp, which are similar to the site-size of the proper (7(±1) nucleotides) and the total (16(±2) nucleotides) DNA-binding site of the ASFV pol X, determined in

77

Figure 2. The primary structures of the dsDNA oligomers, used to examine the interactions with ASFV pol X. The oligomers contain (a) 10, (b) 16, and (c) 20 basepairs. Each oligomer has a coumarin derivative, CP, located at one of its 5′ ends, which provides the spectroscopic signal to monitor interactions with the ASFV pol X.

interactions with the ssDNA. 17 Moreover, the selected dsDNA oligomers allow us to perform titrations over large ranges of the concentration of DNA and protein and avoiding precipitation of the sample. Fluorescence titrations of the CP-labeled dsDNA 10-mer (Figure 2, substrate A) with the ASFV pol X at two different concentrations of nucleic acid, in standard buffer (see Materials and Methods), are shown in Figure 3(a). At a higher concentration of DNA, a given relative fluorescence quenching, ΔF, is reached at higher concentrations of polymerase. This results from the fact that at a higher concentration of DNA, more protein is required to obtain the same total average degree of binding, ∑Θi. The maximum observed quenching is ∼0.6. The selected concentrations of DNA provide the separation of binding isotherms up to ΔF ∼0.43. To obtain thermodynamically rigorous binding parameters, independent of any assumption about the relationship between the observed signal and the total average degree of binding, ∑Θi, titration curves, such as in Figure 3(a), have been analyzed by using the approach outlined in Materials and Methods.17–25,32–39 Figure 3(b) shows the dependence of the observed relative fluorescence quenching, ΔF, as a function of the total average degree of binding, ∑Θi, of the ASFV pol X on the 10-mer. The plot is linear and short extrapolation to the maximum value of the fluorescence quenching, ΔFmax = 0.58 ± 0.05, gives the maximum value of ∑Θi = 0.9 ± 0.1. Thus, at saturation, only a single ASFV pol X molecule binds to the dsDNA 10-mer.17–25,32–39

78

ASFV Pol X-dsDNA Interactions

Figure 3. (a) Fluorescence titrations of the dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 198 mM NaCl and 1 mM MgCl2, at two different concentrations of the nucleic acid, 2 × 10−9 M (■) and 1.5 × 10−8 M (□), respectively. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the single-site model (equation (1)), using a single set of the binding and spectroscopic parameters, K10 = 7.6 × 107 M−1, and ΔFmax = 0.58 (see the text for details). (b) The dependence of the observed relative fluorescence quenching, ΔF, upon the total average degree of binding, ∑Θi, of ASFV pol X on the dsDNA 10-mer. The values of ∑Θi were obtained using the quantitative approach described in Materials and Methods. The continuous line follows the experimental points and does not have a theoretical basis. The broken line is an extrapolation of the plot to the maximum observed fluorescence quenching, ΔFmax = 0.58, marked by the horizontal continuous line. (c) Fluorescence titrations of the dsDNA 20-mer (Figure 2, substrate C) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 198 mM NaCl and 1 mM MgCl2, at two different concentrations of the nucleic acid, 4.8 × 10−9 M (■) and 1.5 × 10−8 M (□), respectively. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the model of cooperative binding of large ligand binding to a short lattice, which can accommodate two ligand molecules (equations (3)–(5)), using a single set of the binding and spectroscopic parameters, K20 = 1.0 × 107 M−1, n = 10, ω = 110, ΔF1 = 0.28, and ΔF2 = 0.103 (see the text for details). (d) The dependence of the observed relative fluorescence quenching, ΔF, upon the total average degree of binding, ∑Θi, of ASFV pol X on the dsDNA 20-mer. The values of ∑Θi (■) were obtained using the quantitative approach described in Materials and Methods. The continuous line is the theoretical dependence of the observed fluorescence quenching of the dsDNA 20-mer, ΔF, upon the total average degree of binding, ∑Θi, of ASFV pol X. The plot was generated using equations (3)–(5), and the binding and spectroscopic parameters obtained for the system.

Fluorescence titrations of the dsDNA 16-mer (Figure 2, substrate B) show the same 1:1 stoichiometry and the corresponding single-phase binding process, as determined for the dsDNA 10-mer (data not shown). However, the situation is very different with the dsDNA 20-mer (Figure 2, substrate C). Fluorescence titrations of the CP-labeled dsDNA 20-mer with the ASFV pol X, at two different concentrations of nucleic acid, are shown in Figure 3(c). The binding process clearly shows two binding phases, differing in the induced changes in the nucleic acid fluorescence. In the high-affinity phase, the binding of the ASFV pol X induces quenching of

the DNA oligomer fluorescence, while in the lowaffinity phase, the quenching decreases indicating an increase in the intensity of the nucleic acid emission. This is an intricate behavior, which is rarely seen in spectroscopic protein–nucleic acid binding studies, where a monotonous increase of the spectroscopic signal is usually observed, even for very complex binding systems. The fact that two binding phases are already observed in the spectroscopic titrations curves already shows that at least two or more ASFV pol X molecules bind to the dsDNA 20-mer. Figure 3(d) shows the dependence of the observed relative

79

ASFV Pol X-dsDNA Interactions

fluorescence quenching ΔF as a function of the total average degree of binding, ∑Θi, of the ASFV pol X on the 20-mer. The maximum quenching of the highaffinity phase is ∼0.15, i.e. it is lower than that observed for the 10-mer (Figure 3(a)). Also, the selected concentrations of DNA provide separation of titration curves up to ΔF ∼0.15. The plot in Figure 3(d) is clearly non-linear. Nevertheless, it undoubtedly shows that only a single molecule of the ASFV pol X binds in the high-affinity phase. We could determine the total average degree of binding up to ∑Θi ∼ 1.3 (Figure 3(d)). To address the maximum stoichiometry of the formed complex, we performed sedimentation equilibrium studies of the molecular mass of the ASFV pol X–dsDNA 20-mer complex, large excess of the protein over the nucleic acid (data not shown). These experiments were facilitated by the fact that we can exclusively monitor the nucleic acid concentration at the absorption band of CP (∼430 nm) in the presence of the concentration of high enzyme. The obtained molecular mass of the complex, at a high concentration of enzyme, is 47,000(±5000) Da, indicating that, at saturation, two ASFV pol X molecules bind to the dsDNA 20-mer (Materials and Methods). Thus, the second binding phase observed in the fluorescence titration curves (Figure 3(c)) reflects the binding of the second polymerase molecule to the nucleic acid (see below). These data and the analysis described below reveal two crucial aspects of ASFV pol X–dsDNA interactions. First, at saturation, only one ASFV pol X molecule binds to the dsDNA 10-mer and 16-mer, while two ASFV pol X molecules bind to the 20-mer. Therefore, the observed behavior indicates strongly that the enzyme forms a single type of complex with the dsDNA and requires no less than 9 bp, optimally 10 bp to form a stable complex with the nucleic acid. This is very different from the ASFV pol X binding to the ssDNA where the site-size of the polymerase– nucleic acid complex is 16(±2) nucleotides.17 Moreover, the obtained intrinsic binding constants, with the examined longer dsDNA oligomers, are similar to the intrinsic binding constants determined for the dsDNA 10-mer, indicating that the similar intrinsic binding process is observed for all examined oligomers. In other words, the obtained data indicate that the site-size of the ASFV pol X–dsDNA complex is n = 10(±2) bp and that there is not a detectable “end effect” on the enzyme binding to the dsDNA. This is in excellent agreement with our previous studies of interactions of the ASFV pol X with the ssDNA

where no end effect in the enzyme binding was observed.17 Second, the maximum stoichiometry of the ASFV pol X binding to the dsDNA 10-mer is not affected by an ∼10-fold increase of the dsDNA oligomer concentration (Figure 3(a)). Such independence of the enzyme–dsDNA stoichiometry upon the short DNA oligomer concentrations provides strong thermodynamic evidence that only one of the two DNA-binding subsites of the ASFV pol X engages in interactions with the dsDNA.25,29,32–39 If the second DNA-binding subsite binds the dsDNA oligomer, its affinity must be at least ∼50-fold lower than the observed affinity. If the affinity were higher, then the ∼10-fold increase of the concentration of 10-mer would detect weak interactions with the second DNA-binding subsite, by showing a dramatic change in the stoichiometry of the complex. This is not observed experimentally. Therefore, the obtained data indicate that the enzyme binds the dsDNA by using only one of its DNA-binding subsites in any of the formed complexes (see Discussion).25,29,32–39 Statistical thermodynamic model of the ASFV Pol X binding to the dsDNA oligomers Binding of the ASFV pol X to the dsDNA 10-mer can be described by a single-site binding isotherm, defined by:
 KN ½PF  ð1Þ DF ¼ DFmax 1 þ KN ½PF  where KN is the intrinsic binding constant of ASFV pol X for the dsDNA 10-mer (N = 10) and [PF] is the concentration of free enzyme. The continuous lines in Figure 3(a) are the non-linear least-squares fits of the experimental spectroscopic titration curves to equation (1) with the binding constant, K10, and maximum fluorescence quenching, ΔFmax, as the fitting parameters (Table 1). The value of the intrinsic binding constant K10 is 7.6(±0.9) × 107 M−1, which is dramatically larger than the analogous parameter K10 ∼1.3 × 105 M−1, previously obtained for the ssDNA conformation, in the same solution conditions. 17 Such a large difference in the affinities between these two nucleic acid conformations indicates that ASFV pol X binds strongly and preferentially to dsDNA over ssDNA (see Discussion).

Table 1. Thermodynamic and spectroscopic parameters of ASFV pol X binding to dsDNA oligomers, containing the coumarin derivative (CP) at the 5′ end of one of the strands of the nucleic acid (Figure 1) and corresponding unlabeled dsDNA oligomers, in buffer C (pH 7.0, 10 °C) (see the text for details) dsDNA oligomer 5′-CP-dsDNA 10-mer (substrate A, Figure 2) dsDNA 10-mer 5′-CP-dsDNA 16-mer (substrate B, Figure 2) dsDNA 16-mer 5′-CP-dsDNA 20-mer (substrate C in Figure 2) dsDNA 20-mer

KN (M−1)

ω

ΔF1

ΔF2

7.6(±1.1) × 107 7.3(±0.9) × 107 6.5(±0.8) × 107 6.7(±0.9) × 107 1.1(±0.2) × 107 1.0(±0.2) × 107

– – – – 110 ± 8 110 ± 8

0.58 ± 0.05

– – – – 0.103 ± 0.005 –

0.35 ± 0.02 0.28 ± 0.01 –

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ASFV Pol X-dsDNA Interactions

Binding of ASFV pol X to the dsDNA 16-mer is described also by the single-site isotherm as:
 ðNn þ 1ÞKN ½PF  DF ¼ DFmax ð2Þ 1 þ ðNn þ 1ÞKN ½PF  where KN is the intrinsic binding constant for the dsDNA 16-mer. The statistical factor N–n + 1 takes into account that there are several potential binding sites for the protein, with the site-size n = 10, on the 16-mer.25,38–42 The value of KN for the 16-mer is K16 = 6.7(±0.9) × 10 7 M −1 , which, within experimental accuracy, is identical with the intrinsic value determined for the 10-mer (Table 1). The simplest statistical thermodynamic model that describes the binding of two ASFV pol X molecules to the dsDNA 20-mer is defined by the partition function ZN, as:25,38–42 ZN ¼ 1 þ ðNn þ 1ÞK½PF  þ NðK½PF Þ2

ð3Þ

where N is the total number of base-pairs in the oligomer (N = 20), n is the site-size of the ASFV pol X–dsDNA complex, and ω is the parameter characterizing the cooperative interactions between the bound protein molecules.25,38–42 The total average degree of binding, ∑Θi, is described by the standard statistical thermodynamic formula, ∂ln ZN/∂ln [PF], as: X ½ðNn þ 1ÞK PF þ 2NðKPF Þ2  Qi ¼ ZN

ð4Þ

The observed relative fluorescence quenching, ΔF, of the dsDNA 20-mer is then: " #   ðNn þ 1ÞK PF NðK PF Þ2 DF ¼ DF1 þ DF2 ð5Þ ZN ZN where ΔF1 is the relative molar fluorescence quenching accompanying the binding of the first and second ASFV pol X molecule to the dsDNA 20-mer, and ΔF2 is the relative fluorescence quenching induced by binding of two polymerase molecules to the nucleic acid. The observed binding process is complex. To address the analysis of the fluorescence titration curves of the dsDNA 20-mer quantitatively, we applied the following strategy, which is possible only because we have both the fluorescence titration curves (Figure 3(c)) and the dependence of the observed quenching, ΔF, as a function of the total average degree of binding, ∑Θi, (Figure 3(d)). The value of ΔF1 can be determined from the initial part of the plot in Figure 3(d) as ΔF1 = ∂ΔF/∂∑Θi, which provides ΔF1 = 0.28 ± 0.01. The value of ΔF2 can be estimated from the plateau of the titration curves at high concentrations of enzyme, which provides ΔF2 = 0.103 ± 0.005. Thus, there are only two remaining parameters that must be determined, K20 and ω. The continuous lines in Figure 3(c) are the non-linear least-squares fits of the experimental titration curves to (2)–(4), with intrinsic binding constant K20 and

cooperativity parameter ω as the fitting parameters. The values of the binding and spectroscopic parameters are included in Table 1. The continuous line in Figure 3(d) is the theoretical dependence of the observed fluorescence quenching, ΔF, as a function of the total average degree of binding, ∑Θi, using the values of K20 and ω. It is clear that the model ((2)–(4)) provides an excellent description of the experimentally observed binding process. The intrinsic binding constant K20 is lower, by a factor of ∼6–7, than the corresponding parameter obtained for the 10-mer and 16-mer. Surprisingly, the value of the cooperativity parameter ω is 110 ± 8, indicating that, unlike the binding to the ssDNA conformation, interactions of ASFV pol X with the dsDNA are characterized by significant positive cooperative interactions (see Discussion). Binding of ASFV Pol X to unmodified dsDNA oligomers Quantitative analysis of ASFV pol X binding to analogous unmodified dsDNA oligomers has been performed using the MCT method outlined in Materials and Methods.25,38 In these studies, we use the dsDNA 10-mer labeled with CP (Figure 2, substrate A), as a reference fluorescent nucleic acid, which provides the highest value of observed fluorescence quenching. Fluorescence titration of the CP-labeled dsDNA 10-mer (2 × 10−9 M) with ASFV pol X in the presence of an unlabeled dsDNA 10-mer (2 × 10−8 M), in standard buffer, is shown in Figure 4(a). The titration curve of the labeled 10-mer alone with the enzyme, at the same reference concentration of nucleic acid as in the titration performed in the presence of an unmodified nucleic acid, is included in Figure 4(a). The presence of the competing, unmodified oligomer shifts the titration curve toward higher concentrations of protein, due to the simultaneous binding of the protein to the fluorescent and the unmodified DNA. Applying the MCT method (Materials and Methods), the degree of binding, (∑Θi)S, and the maximum stoichiometry of the complex formed can then be obtained using (18)–(20), which provides the maximum stoichiometry of one ASFV pol X molecule bound to the unmodified dsDNA 10-mer.25,38 The continuous line in Figure 4(a) is the non-linear least-squares fit of the experimental titration curve for the simultaneous binding of ASFV pol X to two competing dsDNA oligomers, each accommodating a single enzyme molecule. Because the binding constant and the maximum fluorescence quenching for the labeled DNA have been obtained independently (see above), there is only one parameter, the intrinsic binding constant K10, which has to be determined (Table 1). Fluorescence titration of the CP-labeled dsDNA 10-mer (2 × 10−9 M) with ASFV pol X in the presence of unlabeled dsDNA 20-mer (1 × 10−8 M) is shown in Figure 4(b). The titration curve of the labeled oligomer alone with the enzyme, at the same reference concentration of nucleic acid as in the titration per-

ASFV Pol X-dsDNA Interactions

81 DNA. The binding parameters for all unmodified dsDNA oligomers are included in Table 1. It is evident that binding of the enzyme to unmodified dsDNAs is characterized, within experimental accuracy, by the same parameters as binding to the modified DNA (Table 1). In other words, the presence of the CP moiety does not affect the enzyme interactions with the CP-labeled dsDNAs to any detectable extent. Salt effect on the intrinsic affinity of ASFV Pol X–dsDNA 10-mer interactions

Figure 4. (a) Fluorescence titrations of the labeled dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 198 mM NaCl and 1 mM MgCl2, in the absence (■) and in the presence (□) of the corresponding unmodified dsDNA 10-mer. The concentrations of the labeled and unmodified oligomers are 2 × 10−9 M and 2 × 10−8 M (oligomer), respectively. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the single-site model (equation (1)), using a single set of the binding and spectroscopic parameters, K10 = 7.6 × 107 M−1, and ΔFmax = 0.58, for the labeled oligomer and K10 = 7.3 × 107 M−1, for the unmodified nucleic acid. (b) Fluorescence titrations of the labeled dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 198 mM NaCl and 1 mM MgCl2, in the absence (■) and in the presence (□) of the unmodified dsDNA 20-mer. The concentrations of the labeled and unmodified oligomers are 2 × 10−9 M and 1 × 10−8 M (oligomer), respectively. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the model of cooperative binding of large ligand binding to a short lattice, which can accommodate two ligand molecules (equations (3)– (5)), using a single set of binding and spectroscopic parameters, K10 = 7.6 × 107 M−1, and ΔFmax = 0.58, for the labeled 10-mer and K20 = 1.1 × 107 M−1, n = 10, ω = 110, for the unmodified nucleic acid (see the text for details).

formed in the presence of the unmodified nucleic acid, is included in Figure 4(b). The continuous line in Figure 4(b) is the non-linear least-squares fit of the experimental titration curve, using the statistical thermodynamic model (equation (3)) for both nucleic acids.38 In this case, the fit includes two parameters, the intrinsic binding constant K20S and the cooperativity parameter ω, for the unmodified

Fluorescence titrations of the CP-labeled dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X, in standard buffer containing different concentrations of NaCl, are shown in Figure 5(a). Analogous titrations, in the presence of NaBr, are shown in Figure 5(c). The increasing concentration of salt decreases significantly the affinity of the enzyme for the nucleic acid. The maximum fluorescence quenching at saturation ΔFmax also decreases with the increasing concentration of NaCl or NaBr, indicating that the salt affects the affinity and the structure of the enzyme–nucleic acid complex. The continuous lines in Figure 5(a) and (c) are non-linear least-squares fits of the experimental titration curves to equation (1). Figure 5(b) and (d) show the dependence of the logarithm of the ASFV pol X intrinsic binding constant K10 upon the logarithm of the concentrations of NaCl and NaBr (log-log plots).43,44 The plots are linear and characterized by the slopes ∂logK10/∂log [NaCl] = −1 ± 0.3 and ∂logK10/∂log [NaBr] = −2 ± 0.5. Thus, a net release of one or two ions accompanies the intrinsic interaction in the ASFV pol X–dsDNA 10-mer complex, as observed for the complex with the ssDNA.43,44 However, a significantly higher slope observed in the presence of NaBr, i.e. where chloride anions are replaced by bromide, indicates that both cations and anions participate in the ion-exchange process (see Discussion). The salt effect on the intrinsic affinity and cooperativity of the ASFV Pol X–dsDNA 20-mer interactions Correspondingly, we addressed the salt effect on ASFV pol X binding to the dsDNA 20-mer, where the enzyme engages in both the intrinsic and cooperative interactions. Fluorescence titrations of the CP-labeled dsDNA 20-mer (Figure 2, substrate C) with ASFV pol X in standard buffer, containing different concentrations of NaCl, are shown in Figure 6(a). Analogous titrations, in the presence of NaBr, are shown in Figure 6(c). Similar to the effect observed for the 10-mer, increasing the concentration of salt decreases the affinity of the enzyme for the nucleic acid significantly. However, it does not change the biphasic character of the binding process. The analysis of the titration curves has been performed analogously as described above (Figure 3(c)). The continuous lines in Figure 6(a) and (c) are

82

ASFV Pol X-dsDNA Interactions

Figure 5. (a) Fluorescence titrations of the dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0, 10 °C) containing 1 mM MgCl2 and different concentrations of NaCl; (■) 100 mM, (□) 149 mM, (●) 198 mM, (○) 247 mM, (▲) 296 mM, (Δ) 394 mM. The continuous lines are the non-linear least-squares fits of the titration curves to the single-site isotherm (equation (1)) using K10 = 1.5× 108 M−1 and ΔFmax = 0.66 (■), K10 = 8.9 × 107 M−1 and ΔFmax = 0.615 (□), K10 = 7.6 × 107 M−1 and ΔFmax = 0.58 (●), K10 = 5.9 × 107 M−1 and ΔFmax = 0.307 (○), K10 = 5.1 × 107 M−1 and ΔFmax = 0.165 (▲), K10 = 4 × 107 M−1 and ΔFmax = 0.115 (Δ). The concentration of the dsDNA oligomer is 1.5 × 10−8 M. (b) The dependence of the logarithm of the intrinsic binding constants K10 (■) upon the logarithm of the concentration of NaCl. The continuous line is the linear least-squares fit, which provides the slope, ∂logK10/∂log [NaCl]= −1.0 ± 0.3. (c) Fluorescence titrations of the dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 1 mM MgCl2 and different concentrations of NaBr; (■) 100 mM, (□) 149 mM, (●) 198 mM, (○) 247 mM, (▲) 296 mM. The continuous lines are the nonlinear least-squares fits of the experimental titration curves to the single-site isotherm (equation (1)), using K10 = 2.3 × 108 M−1 and ΔFmax = 0.36 (■), K10 = 1.1 × 108 M−1 and ΔFmax = 0.34 (□), K10 = 5.6 × 107 M−1 and ΔFmax = 0.31 (●), K10 = 4 × 107 M−1 and ΔFmax = 0.25 (○), K10 = 2.7 × 107 M−1 and ΔFmax = 0.2 (▲). The concentrations of the DNA substrate are 1.5 × 10−8 M. (d) The dependence of the logarithm of the intrinsic binding constants K10 (■) upon the logarithm of the concentration of NaBr. The continuous line is the linear least-squares fit, which provides the slope ∂logK10/∂log [NaBr] = −2.0 ± 0.5.

non-linear least-squares fits of the experimental titration curves to equations (3)–(6). The binding and spectroscopic parameters are included in the Figure legends. Figure 6(b) and (d) show the dependence of the logarithm of the ASFV pol X intrinsic binding constant K20 upon the logarithm of the concentrations of NaCl and NaBr.43,44 The plots are linear and are characterized by the slopes ∂logK/∂log [NaCl] = −1.1 ± 0.3 and ∂logK/∂log [NaBr] = −2 ± 0.5, respectively. Thus, a net release of one or two ions accompanies the intrinsic interaction between the enzyme and the dsDNA 20-mer. First, these values are, within experimental accuracy, the same as the corresponding parameters obtained for the 10-mer (Figure 5(b) and (d)). Second, they are very different from the salt effect on the corresponding ssDNA 20-mer, where the net release of 5.9(±0.3) ions accompanies ASFV pol X binding to the 20-mer

(see Discussion).17 The analogous log-log plots of the dependence of the logarithm of the cooperativity parameter ω upon the logarithm of the concentrations of NaCl and NaBr are, within experimental accuracy, linear and characterized by slopes ∂logω/∂log [NaCl] = −0.6 ± 0.2 and ∂logω/∂log [NaBr] = −1 ± 0.3, respectively (data not shown). Thus, the cooperative interactions are accompanied by a net ion release, unlike what happens in the presence of different anions (see Discussion). Temperature effect on the intrinsic ASFV Pol X–dsDNA 10-mer interactions To further address the nature of the intrinsic ASFV pol X–dsDNA interactions, we examined the temperature effect on the enzyme binding to the dsDNA 10-mer. Fluorescence titrations of the CP-labeled

ASFV Pol X-dsDNA Interactions

83

Figure 6. (a) Fluorescence titrations of the dsDNA 20-mer (Figure 2, substrate C) with ASFV pol X in buffer C (pH 7.0, 10 °C), containing 1 mM MgCl2 and different concentrations of NaCl; (■) 149 mM, (□) 198 mM, (●) 225 mM, (○) 247 mM. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the short lattice model, which can accommodate two ligand molecules (equations (3)–(5)), using K20 = 1.3 × 107 M−1, ω = 130, ΔF1 = 0.33, and ΔF2 = 0.123 (■), K20 = 1 × 107 M−1, ω = 110, ΔF1 = 0.28, and ΔF2 = 0.103 (□), K20 = 8.5 × 106 M−1, ω = 103, ΔF1 = 0.23, and ΔF2 = 0.06 (●), K20 = 7.5 × 106 M−1, ω = 91, ΔF1 = 0.155, and ΔF2 = 0.103 (○). The concentrations of the DNA substrate are 1.5 × 10−8 M. (b) The dependence of the logarithm of the intrinsic binding constants K20 (■) upon the logarithm of the concentration of NaCl. The continuous line is the linear least-squares fit, which provides the slope, ∂logK20/∂log [NaCl] = −1.1 ± 0.3. (c) Fluorescence titrations of the dsDNA 20-mer (Figure 2, substrate C) with ASFV pol X in buffer C (pH 7.0, 10 °C) containing 1 mM MgCl2 and different concentrations of NaBr; (■) 149 mM, (□) 198 mM, (●) 225 mM, (○) 247 mM. The continuous lines are the non-linear least-squares fits of the experimental titration curves to the short lattice model, which can accommodate two ligand molecules (equations (3)–(5)), using: K20 = 1 × 107 M−1, ω = 120, ΔF1 = 0.39, and ΔF2 = 0.123 (■), K20 = 6 × 106 M−1, ω = 100, ΔF1 = 0.29, and ΔF2 = 0.07 (□), K20 = 5 × 106 M−1, ω = 83, ΔF1 = 0.2, and ΔF2 = 0.05 (●), K20 = 3.8 × 106 M−1, ω = 75, ΔF1 = 0.15, and ΔF2 = 0.03 (○). The concentrations of the DNA substrate are 1.5 × 10−8 M. (d) The dependence of the logarithm of the intrinsic binding constants K20 (■) upon the logarithm of the concentration of NaCl. The continuous line is the linear least-squares fit, which provides the slope ∂logK20/∂log [NaBr] = −2.0 ± 0.5.

dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X, in standard buffer, at different temperatures, are shown in Figure 7(a) and (b). The behavior of this seemingly simple system is very complex. At the lowest temperatures examined, in the range from 2.5 °C to 10 °C, the maximum relative fluorescence quenching ΔFmax of the nucleic acid fluorescence increases with temperature (Figure 7(a)). However, above ∼10 °C, the titration curves are characterized by lower and lower values of ΔFmax with increased temperature (Figure 7(b)). For comparison, the titration, performed at 10 °C, is included in Figure 7(a) and (b). Because in the temperature range examined (2.5 °C to 30 °C), the dsDNA completely preserves its double helical structure (Tm ≈ 54 °C) (Materials and Methods), these data provide a strong indication that the structure of the

enzyme undergoes significant changes as the temperature of the sample increases. The continuous lines in Figure 7(a) and (b) are non-linear least-squares fits of the experimental titration curves to a single-site isotherm (equation (10), with the intrinsic binding constant K10 and ΔFmax as fitting parameters. Figure 7(c) shows the dependence of the natural logarithm of the ASFV pol X intrinsic binding constant K10 upon the reciprocal of temperature (Kelvin) (Arrhenius plot).45 The plot is clearly non-linear. The value of K10 has an apparent plateau in the low temperature range. Subsequently, K10 decreases and reaches another plateau above ∼10 °C (Figure 7(c)). Thus, below ∼5 °C, the intrinsic ASFV pol X–dsDNA 10-mer interactions are independent of the temperature, weaken in the temperature range between ∼5 °C and 10 °C and, once again, become tempera-

84

ASFV Pol X-dsDNA Interactions

ture-independent above ∼10 °C. As mentioned above, in the entire temperature range studied, the dsDNA 10-mer fully preserves its structure.46 Thus, the observed non-linear Arrhenius plot in Figure 7(c) indicates strongly that there are two temperatureindependent binding processes in the dsDNA 10-mer association with the ASFV pol X, differing in intrinsic affinities by a factor of ∼4.5 and separated by the temperature-dependent phase. The simplest model, which can account for the observed non-linear behavior of the Arrhenius plot in Figure 7(c), includes a conformational transition of ASFV pol X between two conformations and binding of the dsDNA 10-mer to each conformation with different intrinsic affinities. The model is described by the set of equilibrium reactions: P1 X P2

ð6Þ

P1 þ NF X C1

ð7Þ

P2 þ NF X C2

ð8Þ

where P1 is the conformation of ASFV pol X in the low temperature range, P2 is the corresponding conformation of the enzyme in the high temperature range, C1 and C2 are complexes of the dsDNA 10mer with the low-temperature and high-temperature conformation of the polymerase, respectively. The equilibrium transition, P1 b–N P2, is characterized by the equilibrium constant KC while the binding of the dsDNA 10-mer to P1 and P2 is described by the intrinsic binding constants K1 and K2, respectively. The experimentally observed overall binding constant Kov is then defined by: Kov ¼

Figure 7. (a) Fluorescence titrations of the dsDNA 10-mer (Figure 2, substrate A) with ASFV pol X in buffer C (pH 7.0), containing 1 mM MgCl2 at different temperatures below 10 °C; (Δ) 2.5 °C, (■) 5 °C, (□) 7.5 °C, (•) 8 °C, (○) 9 °C, and (♦) 10 °C. (b) Analogous fluorescence titrations of the dsDNA 10-mer with ASFV pol X in buffer C (pH 7.0), containing 1 mM MgCl2 at different temperatures above 10 °C; (□) 15 °C, (■) 20 °C, (○) 25 °C, (Δ) 30 °C. For comparison, the titration, performed at 10 °C, (♦), is included in both panels. The continuous lines are the nonlinear least-squares fits of the experimental titration curves to the single-site binding isotherm (equation (1)). The concentration of the DNA substrate is 1.5 × 10−8 M. (c) The dependence of the natural logarithm of the intrinsic binding constant K10 (■) upon the reciprocal of the temperature (Kelvin) (Arrhenius plot). The continuous line is the non-linear least-squares fit of the experimental plot to the model of the nucleic acid binding to two ASFV pol X conformations, defined by equations (6)–(12) (see the text for details).

C1 þ C2 K1 þ K2 KC ¼ ðP1 þ P2 ÞNF 1 þ KC

ð9Þ

The temperature-dependence of the particular equilibrium constants in equation (9) are described by: 
 DH1 1 1  ð10Þ K1 ¼ K1 ðTR Þexp  R T TR 
 DH2 1 1  K2 ¼ K2 ðTR Þexp  ð11Þ R T TR 
 DHC 1 1  KC ¼ KC ðTR Þexp  ð12Þ T TR R where K1(TR) and K2(TR), are the intrinsic binding constants of the nucleic acid for the P1 and P2 conformations of the polymerase at a reference temperature, TR, KC(TR) is the equilibrium constant characterizing the enzyme conformational transition at the reference temperature TR, ΔH1 and ΔH2 are the enthalpy changes characterizing the dsDNA 10-mer binding to the P1 and P2 conformation, respectively. ΔHC is the enthalpy change characterizing the conformational transition P1 b–N P2, of the ASFV pol X. The reference temperature TR is taken

ASFV Pol X-dsDNA Interactions

85

as the lowest experimentally studied temperature, i.e. 2.5 °C. There are six parameters, K1(TR), K2(TR), KC(TR), ΔH1, ΔH2, and ΔHC in equation (9). However, the values of several of them can be estimated from the plot in Figure 7(c). The plateaus of the plot indicate that ΔH1 = ΔH2 = 0 and the values of K1(TR) and K2(TR) are equal to the values of these binding constants at low and high temperature plateaus; i.e. 3.1(±0.5) × 108 M−1and 7.6(±0.9) × 107 M−1, respectively. Thus, there are only two unknown parameters in, KC(TR) and ΔHC. The continuous line in Figure 7(c) is the non-linear least-squares fit of the plot to (8)–(11), which provides KC(TR) = 0.05 ± 0.015 and ΔHC = 110(±50) kcal/mol. Although there is a significant error in these parameters, nevertheless, the value of ΔHC clearly indicates that the ASFV pol X undergoes a dramatic structural transition, characterized by a large enthalpy change. The value of KC(TR) indicates that below ∼5 °C, the polymerase exists predominantly in the P1 state. On the other hand, introducing the value of ΔHC into equation (12), gives the value of KC larger than the value of ∼10 already at 10 °C, indicating that the enzyme is predominantly in the P2 state in the high-temperature region (see Discussion). Topology of the ASFV Pol X–dsDNA complex To address the topology of the ASFV pol X– dsDNA complex, we performed fluorescence energy transfer measurements of the enzyme complex with the dsDNA 10-mer, which binds only to the proper DNA-binding-subsite of the polymerase (Table 1).23,29,47–49 The fluorescence energy transfer is the method of choice for determining the structure of large complexes in solution.50–54 We utilize the fact that ASFV pol X has a single tryptophan residue, W92, located on the surface of the N-terminal catalytic domain.15,16 This residue can serve as a single fluorescence energy transfer donor to an acceptor located on the dsDNA oligomer. Thus, measurements of the fluorescence energy transfer efficiency will provide unique information about the location of the bound dsDNA oligomer, with respect to the N-terminal catalytic domain of the enzyme.23,29 The series of DNA 10-mers, which we selected for these studies, is depicted in Figure 8.29 The oligomers have the same primary structure as the dsDNA 10mer used in the thermodynamic studies described above. Each DNA substrate has a fluorescent label, 7-methoxy-coumarin (CPM), attached through a six carbon linker at the 5′ or 3′ terminus of one of the ssDNA strands. Thus, there are four possible locations for the CPM residue on the dsDNA oligomer, with two locations at the 5′ and 3′ ends, respectively (Figure 8). The binding parameters of the enzyme to the CPM-labeled 10-mers are indistinguishable from the binding parameters obtained for the CP-labeled DNA substrates or unmodified DNA (data not shown). Unlike the CP derivative used in the binding studies (see above), CPM has an absorption maximum at ∼355 nm,

Figure 8. The dsDNA oligomers used in the fluorescence energy transfer studies of the topology of the ASFV pol X–dsDNA complex. In (a) and (b), the dsDNA 10-mer has a coumarin derivative, CPM, attached to the 5′ end of one the ssDNA strands. In the 10-mers, (c) and (d), the CPM derivative is attached to the 3′ end of one of the ssDNA strands (Materials and Methods).

which strongly overlaps the tryptophan fluorescence spectrum.29,32,49 Such an overlap of the acceptor absorption spectrum with the donor emission spectrum is a condition for the fluorescence resonance energy transfer to occur.23,29,47–54 Using the emission spectrum of ASFV pol X and the absorption spectra of the labeled 10-mers, we determined the Förster critical distance Ro for the CPM on the dsDNA and ASFV pol X tryptophan as 21.7 Å (data not shown).29 Fluorescence emission spectra (λex = 295 nm) of the ASFV pol X complex with the unlabeled dsDNA 10-mer and with the dsDNA 10-mer, labeled at the 5′ end with CPM (Figure 8, substrate A), and the emission spectrum of the same CPM-labeled DNA in the absence of the protein (λex = 295 nm), in standard buffer, are shown in Figure 9(a). The total concentrations of the polymerase and the nucleic acid are 2 × 10−7 M and 5 × 10−8 M, respectively. The spectra show that the presence of the acceptor (CPM) induces significant quenching of the donor fluorescence. Correspondingly, there is an increase of the acceptor fluorescence emission in the presence of the donor. Both features, the donor emission quenching and the sensitized acceptor emission, indicate that an efficient fluorescence energy transfer

86

ASFV Pol X-dsDNA Interactions

Figure 9. (a) Fluorescence emission spectrum of ASFV pol X alone (__ __), the ASFV pol X–dsDNA oligomer complex (_____) and the 5′-CPM-dsDNA oligomer (– – –) (Figure 8, substrate A) (λex = 295 nm) in buffer C (pH 7.0, 10 °C) containing 198 mM NaCl and 1 mM MgCl2; the normalized emission spectrum of ASFV pol X to the maximum of its emission at 343 nm in the complex with the dsDNA oligomer (– – –); the emission spectrum of the 5′-CPM-dsDNA oligomer in the complex with the enzyme (__ _ _ _ __). (b) Fluorescence emission spectrum of ASFV pol X alone (__ __), the ASFV pol X - dsDNA oligomer complex (_____), and the 5′-CPM-dsDNA oligomer (– – –), (Figure 8, substrate B) (λex = 295 nm) in buffer C (pH 7.0, 10 °C) containing 198 mM NaCl and 1 mM MgCl2; the normalized emission spectrum of ASFV pol X to the maximum of its emission at 343 nm in the complex with the dsDNA 10-mer (_ _ _); the emission spectrum of the 5′-CPM-dsDNA oligomer in the complex with the enzyme (__ _ _ _ __). (c) Fluorescence emission spectrum of ASFV pol X alone (__ __), the ASFV pol X - dsDNA oligomer complex (_____), and the 3′-CPMdsDNA oligomer (– – –), (Figure 8, substrate C) (λex = 295 nm), in buffer C (pH 7.0, 10 °C) containing 198 mM NaCl and 1 mM MgCl2; the normalized emission spectrum of ASFV pol X to the maximum of its emission at 343 nm in the complex with the oligomer (_ _ _); the emission spectrum of the 3′-CPM-dsDNA oligomer in the complex with the enzyme (__ _ _ _ __). (d) Fluorescence emission spectrum of ASFV pol X alone (__ __), the ASFV pol X - dsDNA oligomer complex (_____), and the 3′-CPM-dsDNA oligomer (– – –), (Figure 8, substrate D) (λex = 295 nm) in buffer C (pH 7.0, 10 °C) containing 198 mM NaCl and 1 mM MgCl2; the normalized emission spectrum of ASFV pol X to the maximum of its emission at 343 nm in complex with the oligomer (_ _ _); the emission spectrum of the 3′-CPM-dsDNA 10-mer in complex with the enzyme (__ _ _ _ __). Concentrations of ASFV pol X and the dsDNA 10-mer in all data presented in (a)–(d) are 2 × 10−7 M and 5 × 10−8 M.

process occurs in the examined ASFV pol X–dsDNA 10-mer system.23,29,47–54 The emission spectrum of the tryptophan donor, in the complex with the CPM acceptor was obtained by normalizing the peak of the donor emission spectrum, recorded in the absence of the acceptor, to the intensity of the same tryptophan donor recorded in the presence of the acceptor. Subsequently, the emission spectrum of the CPM acceptor, in the complex with the donor, has been obtained by subtracting the normalized emission spectrum of the donor from the emission spectrum of the complex, containing both the donor and the acceptor. The normalized emission spectrum of the tryptophan donor in the complex with

the CPM acceptor and the emission spectrum of the CPM acceptor in the complex with the tryptophan donor are included in Figure 9(a). The same measurements and analyses have been performed for the dsDNA 10-mer, containing the CPM marker at the opposite 5′ end (Figure 8, substrate B) and the corresponding spectra are shown in Figure 9(b). The emission intensities of the donor, FD, and the acceptor, FA, alone, and in the complex with the acceptor, FDA, and donor, FAD, respectively, have been obtained by integrating the corresponding emission spectra in Figure 9(a) and (b). At the selected total concentrations of ASFV pol X and the dsDNA 10-mer, the degree of binding of the dsDNA

87

ASFV Pol X-dsDNA Interactions

oligomer to the polymerase and the degree of saturation of the nucleic acid with the enzyme, are νD ≈ 0.23 and, νA ≈ 0.93, respectively, obtained using the binding constant, K = 7.6(±0.9) × 107 M−1, characterizing the binding of the dsDNA 10-mers to ASFV pol X (Table 1). The apparent fluorescence energy transfer efficiencies, ED and EA, Förster fluorescence energy transfer efficiency, E, and the average distance between the donor and acceptor, R (2/3), have then been calculated using equations (13)–(17). The values of all fluorescence energy transfer parameters for the two dsDNA 10-mers labeled at the 5′ end with CPM (Figure 8, substrates A and B) are included in Table 2. The values of the average Förster fluorescence energy transfer efficiency, E, are very similar, ∼0.17 and ∼0.22, respectively, for both oligomers containing the CPM residue at the 5′ ends. As a result, the average distances, R(2/3), are ∼28.3 Å and ∼26.8 Å, also very close to each other. The distance between the 5′ end of the bound dsDNA 10-mer, averaged over these two measurements is ∼27.6 Å. Notice, this distance is longer than the ∼20 Å between W92 and the conserved aspartate residues in the catalytic site of the NMR structure of the enzyme (Figure 1) (see Discussion). Analogous fluorescence energy transfer measurements and analyses have been performed for the ASFV pol X complexes with the dsDNA 10-mers, containing the CPM moiety at the 3′ ends of the nucleic acid (Figure 8, substrates C and D). The corresponding spectra are shown in Figure 9(c) and (d). The difference between the spectra obtained for the oligomers containing the fluorescence energy transfer acceptor at the 5′ end and the oligomers containing the acceptor at the 3′ end is striking. There is very little, if any, quenching of the donor (W92) fluorescence in the presence of the acceptor, CPM, as well as hardly any measurable sensitized emission of the acceptor for both DNA substrates (Figure 9(c) and (d)). Because the error in the measurement of the fluorescence energy transfer efficiency is ∼0.03 in these experiments, the data indicate that the Förster fluorescence energy transfer efficiency E must be ∼0.03 or less (Table 2). Using this conservative estimate of E, one obtains that the average distance between the 3′ end of the bound dsDNA 10-mer and W92 in the catalytic domain must be ≥ 39 Å (Table 2). In other words, it must be close to or larger than twice the length of the Förster

critical distance, Ro = 21.7 Å, of the examined donor– acceptor pair (see Discussion).23,29,47–54 As we pointed out above, the distances between the 5′ and 3′ ends of the bound dsDNA oligomers and W92 have been obtained for two different DNA substrates, each having different locations and environments around the acceptor.23,29 This effectively excludes the possibility that the observed Förster energy transfer efficiency E is a consequence of a very peculiar orientation of the donor and the acceptor, and/or their immobilization, that would result in κ2 = 0 or 4.23,29,55,56 Nevertheless, we have determined the limiting anisotropies of the ASFV pol X and CPM on the bound DNA oligomers to assess the mobility of the donor and the acceptor on the time-scale of their fluorescence lifetimes. The values of the limiting anisotropies for W92 and the CPM on the dsDNA oligomers are 0.18 ± 0.01 and 0.18 ± 0.01, respectively. Thus, they are significantly lower than the fundamental anisotropies of the tryptophan (∼0.27) and, particularly, CPM (∼0.38), at the selected excitation wavelength, indicating that the donor and the acceptor possess significant rotational mobility on the time-scale of their fluorescence lifetimes.29,57 These data additionally indicate that E and the corresponding distances are not affected significantly by any peculiar orientation of the donor and acceptor (Materials and Methods).

Discussion The site-size of the ASFV Pol X–dsDNA DNA-binding subsites of the polymerase engages in interactions with the dsDNA Studies with ssDNA revealed that the total DNAbinding site of ASFV pol X has a heterogeneous structure and contains two nucleic acid-binding regions.17 The region with high nucleic acid affinity was identified as the proper DNA-binding site and occludes only 7(±1) nucleotides. However, the enzyme has a second DNA-binding area characterized by a much lower nucleic acid affinity, which encompasses a similar seven to eight nucleotides. Consequently, the total site-size of the ASFV pol X–ssDNA complex is 16(±2) nucleotides. The presence of the two nucleic acid-binding regions corroborates well with the NMR structure of the protein (Figure 1), where two lysine-rich regions of

Table 2. Fluorescence energy transfer parameters for the single donor, tryptophan residue, W92, of the ASFV pol X and the single acceptor, CPM moiety at the 5′ or 3′ end of the dsDNA 10-mer (Figure 8), in buffer C (pH 7.0, 10 °C) (see the text for details) dsDNA 10-mer A B C D

ED

EA

E

Ro (Å)

R(2/3) (Å)

R(2/3)av (Å)a

0.67 ± 0.03 0.54 ± 0.03 ≤0.03 ≤0.03

0.07 ± 0.03 0.22 ± 0.03 ≤0.03 ≤0.03

0.17 ± 0.02 0.22 ± 0.03 ≤0.03 ≤0.03

21.7 21.7 21.7 21.7

28.3 ± 2.1 26.8 ± 2.1 ≥39 ≥39

27.6 ± 2.1 27.6 ± 2.1 ≥39 ≥39

a Averaged over two measurements for two locations of the fluorescence energy transfer acceptor (CPM) at the 5′ or 3′ end (see the text for details).

88 the protein have been proposed as the potential areas of interaction.15,16 However, the NMR structure alone does not provide any clue about the reason for the dramatic difference in the intrinsic affinities between these two areas discovered in thermodynamic studies.17 Similar heterogeneity of the total DNA-binding site has been found for the mammalian pol β.23,24,29–31,58,59 As a result, pol β forms different binding modes with the ssDNA, where the enzyme occludes different numbers of nucleotides by engaging one or both DNA-binding subsites. However, ASFV pol X does not form different binding modes with ssDNA, an indication that the DNA-binding subsites of the enzyme are much less autonomous than the analogous subsites of pol β.17 In other words, the ASFV pol X DNA-binding subsites seem to act as a single entity in interactions with the ssDNA. In this context, the small site-size of 10(±2) bp of the ASFV pol X–dsDNA complex, as compared to its total site-size of 16(±2) nt with the ssDNA, is very surprising. This small site-size is comparable to the site-size of 7(±1) nt of the identified proper DNAbinding site of the enzyme. Thus, the thermodynamic data provide the first evidence that ASFV pol X binds the dsDNA using only one of the DNAbinding subsites in the complexes formed. As we pointed out, the independence of the ASFV pol X–dsDNA stoichiometry upon the concentration of the DNA oligomer indicates that one of the DNAbinding subsites is incapable of engaging in interactions with the dsDNA. Moreover, the high dsDNAaffinity of the subsite, which is engaging in interactions with the dsDNA, indicates that this is the proper DNA-binding subsite.23,24,29–31,58,59 ASFV pol X binds dsDNA exclusively using its non-catalytic C-terminal domain: the location of the proper DNA-binding subsite of the enzyme As pointed out above, the NMR structure alone does not provide any indication as to which of the lysine-rich regions serves as the proper DNAbinding site, i.e. it is used exclusively by ASFV pol X in binding to the dsDNA. On the other hand, the location of the proper DNA-binding subsite, within the total DNA-binding site, is indicated by the fluorescence energy transfer data, using four DNA 10-mers with the fluorescence acceptor placed at the 5′ or 3′ end of the nucleic acid (Figure 8). When the acceptor is at one of the 5′ ends of the nucleic acid, the average fluorescence energy transfer E is ∼ 0.19, which corresponds to the average distance between the 5′ end of the bound DNA and W92 in the catalytic domain, R(2/3) ∼ 27.6 Å (Table 2). Conversely, when the acceptor is at one of the 3′ ends of the bound 10-mer, the average value of E is ≤0.03, which indicates that the average distance between the 3′ ends of the bound nucleic acid and the tryptophan residue must be larger than 39 Å. The plausible model of the observed behavior is shown schematically in Figure 10(a) and (b). The catalytic site of the polymerase contains three

ASFV Pol X-dsDNA Interactions

conserved aspartate residues, D49, D50, and D101, and the location of the catalytic site is marked in Figure 10(a) and (b). In Figure 10(a), the dsDNA 10mer binds to the DNA-binding subsite on the noncatalytic C-terminal domain. The 10-mer containing the CPM acceptor at one of its 5′ ends can bind to the subsite in two equivalent orientations. However, to preserve the same orientation of the dsDNA, as in the catalytic complex with the gapped DNA, in these two possible orientations, the 3′ end of one strand must always be in, or close to, the catalytic site, which is ∼20 Å from W92, while the 5′ end of the opposite strand is always much closer than the 3′ end to W92, by ∼ 20 Å. At the opposite end of the bound dsDNA 10-mer, both the 5′ and 3′ ends are at a similar distance from the W92 residues on the catalytic domain. Taking into account the length of the labeled dsDNA 10-mer, ∼ 38 Å, and the geometry of the NMR structure of ASFV pol X, this distance can be estimated conservatively as 45–50 Å. In other words, when the dsDNA 10-mer is bound to the non-catalytic C-terminal domain, the average distance between its 5′ ends and the single tryptophan residue of the enzyme, W92, will be significantly shorter than the average distance between the 3′ ends of the bound nucleic acid and W92. This is exactly what is observed experimentally (Figure 9(a) and (b); Table 2). The situation is very different if the dsDNA 10mer were bound to the N-terminal catalytic domain. As mentioned above, the CPM-labeled nucleic acid can also bind the N-terminal catalytic domain in two equivalent orientations. However, to preserve the same orientation as in the catalytic complex with the gapped DNA, in each of these two possible orientations, the 3′ end of the bound 10-mer is now closer to W92, while the corresponding 5′ end on the opposite strand must be in, or close to, the catalytic site of the enzyme, approximately, 20 Å from W92. The distance from the opposite end of the bound nucleic acid to W92 is similar for both the 5′ and 3′ ends, and can be estimated conservatively as ∼ 45–50 Å. If this were the case, then the average distance from the 5′ ends to W92 would be larger than the average distance from the 3′ ends of the bound dsDNA 10-mer. However, this is not observed experimentally. The striking difference between the energy transfer efficiencies of two locations of the acceptor, CPM, indicates clearly that the 3′ end is always at a larger average distance from W92 than the 5′ end (Table 2). Therefore, the fluorescence energy transfer data provide strong evidence that the dsDNA 10-mer is bound to the C terminus of ASFV pol X, i.e. the proper DNAbinding subsite is located on the C-terminal noncatalytic domain of the enzyme. Notice, the distance from W92 and the aspartate residues in the catalytic site of ASFV pol X in the NMR structure of the enzyme is ∼ 20 Å (Figure 1). The obtained average distance from the 5′ end of the dsDNA 10-mer, bound to the C-terminal noncatalytic domain, to the same tryptophan residue is ∼27.6 Å (Table 2). However, the measured distance

ASFV Pol X-dsDNA Interactions

is the average for the two possible equivalent orientations of the bound 10-mer and, in one of these orientations, the 5′ end can be as far from the tryptophan residue as ∼50 Å (see above). With this conservative estimate, the measured average distance between the 5′ end and W92, ∼27.6 Å, places the 5′ end of the bound 10-mer at ∼ 5 Å from the tryptophan residue. On the other hand, the 3′ end of the labeled 10-mer is placed either in, or close to, the catalytic site of the enzyme, i.e. at a distance of ∼20 Å from W92, or at a distance of ∼50 Å in the equivalent orientation. Therefore, the distance between the 3′ end and W92, averaged over the two equivalent orientations of the bound dsDNA 10-mer, is ∼ 35 Å, close to ∼39 Å, estimated on the basis of the fluorescence energy transfer measurements (Table 2). In the context of the discussion above, one can argue that the dsDNA oligomer can rotate freely along its helix axis in the binding site, assuming a multitude of different states. An unlimited rotation, although rather improbable in a binding site that has a defined stoichiometry (site-size) and is geared specifically to bind the dsDNA conformation, is, nevertheless, theoretically possible. However, such a

89 rotation of the dsDNA oligomer would eliminate any difference between the fluorescence energy transfer efficiencies from the donor, W92, to the acceptors located on the 5′ or 3′ ends of the bound nucleic acid, i.e. the locations of the acceptor on the 5′ and the 3′ ends would be completely equivalent in the fluorescence energy transfer experiments. Once again, this equivalence is not observed experimentally. In contrast, the difference between the experimentally observed values of E for the acceptor located at the 5′ end as compared to the acceptor located at the 3′ end is dramatic (Figure 9; Table 2). In other words, the observed difference between the values of E for the acceptor located at the 5′ and 3′ ends of the bound dsDNA oligomer argues strongly against any significant rotation of the dsDNA oligomer bound in the binding site of ASFV pol X. The effect of salt on the intrinsic affinity of the ASFV Pol X–dsDNA complex indicates that only the proper DNA-binding subsite engages in interactions with the nucleic acid Additional evidence for the proposed involvement of only the non-catalytic C-terminal domain in interactions with the dsDNA comes from the salt effect on the observed intrinsic affinities of the ASFV pol X complex with the dsDNA 10-mer (Figure 5(b) Figure 10. (a) A representation of two possible complexes resulting from the association of the dsDNA 10-mer with the DNA-binding subsite located on the non-catalytic C-terminal domain and (b) with the DNA-binding subsite located on the catalytic N-terminus domain of the ASFV pol X. The catalytic site of the polymerase, containing three conserved aspartate residues, D49, D50, and D101 and the location of the tryptophan residue W92 are included in (a) and (b). The 10-mer can bind to each subsite in two equivalent orientations and still preserve the same orientation as in the catalytic complex with the gapped DNA. In (a), the dsDNA 10-mer is bound to the DNAbinding subsite on the C-terminal domain. In each of the two possible orientations, the 3′ end of one strand must always be in, or close to, the catalytic site, which is ∼20 Å from W92, while the 5′ end of the opposite strand is always much closer, by ∼20 Å, to W92. At the opposite end of the bound dsDNA 10-mer, both the 5′ and 3′ ends are at a similar distance from the W92 residues. Thus, when the dsDNA 10-mer is bound to the non-catalytic C-terminal domain, the average distance between any of its 5′ ends and the single tryptophan residue of the enzyme, W92, will be significantly shorter than the average distance between the 3′ end of the bound nucleic acid and the W92, which is in excellent agreement with the fluorescence energy transfer data. In (b), the dsDNA 10-mer is bound to the catalytic N-terminal domain. In each of the two possible equivalent orientations, the 3′ end of the bound 10-mer is closer to W92, while the corresponding 5′ end, on the opposite strand, must be in, or close to, the catalytic site of the enzyme, i.e. at ∼20 Å from W92. The distance from the opposite end of the bound nucleic acid to W92 is similar for both the 5′ and 3′ ends. In such a complex, the average distance from the 5′ ends of the bound dsDNA 10-mer to W92 would be larger than the average distance from the 3′ ends of the bound dsDNA 10-mer. However, this is not observed experimentally (see the text for details).

90 and (d)) and 20-mer (Figure 6(a) and (b)). The same thermodynamic response of the intrinsic affinities of the 10-mer and 20-mer to the changes of the concentration of salt in solution (the same slopes of the log-log plots) indicates that, in the complex with the dsDNA 20-mer, the enzyme does not engage any additional fragment of the nucleic acid, as compared to the complex with the 10-mer. Thus, in both complexes with these different dsDNA oligomers, a very similar intrinsic binding process between the proper DNA-binding subsite, located on the C-terminal domain, is observed, as indicated by similar, although not identical, intrinsic binding constants K10 and K20 (Table 1) (see above). This is a very different behavior from the analogous complexes with the ssDNA 10-mer and 20-mer, where the slopes of the log-log plots for the 10-mer and 20-mer are, in the absence of magnesium, ∂logK10/∂log [NaCl] = −1.3 ± 0.3 and ∂logK 20 /∂log [NaCl] = −5.9 ± 0.4, respectively.17 Such a dramatic difference in the net number of ions released shows clearly that an additional binding area of the protein, beyond the proper DNA-binding subsite, becomes engaged in interactions with the ssDNA 20-mer, as compared to the ssDNA 10-mer.17 On the other hand, the slope ∂logK10/∂log [NaCl] ∼ −1, observed for the dsDNA 10-mer, is very similar to ∂logK10/∂log [NaCl] ∼ −1.3, observed for the analogous complex with the ssDNA 10-mer.17 Although the presence of magnesium will diminish the value of the log-log plot slope, due to the competition with sodium cations, such similar values of the net ion release in the complex formation between the double-stranded and single-stranded conformation indicates a similar character of the observed interactions, i.e. the engagement of the same nucleic acid-binding subsite with the 10-mer. The value of the slope, ∂logK10/∂log [NaBr] ≈ −2, indicating that the net release of approximately two ions accompanies the formation of the complex with the dsDNA in the presence of Br−. Bromide anions are known to have significantly higher affinity for protein amine groups than Cl−.60 The disparity between the slopes obtained in the presence of NaCl and NaBr indicates that the net number of ion release accompanying the intrinsic binding of ASFV pol X to dsDNA is affected by both cation and anion exchanges. Because anions must bind to the protein and not to the DNA, a higher net number of ion released in the presence of NaBr, shows that additional anions are released from the polymerase, when chloride anions is replaced by Br−. The complex salt effect suggests strongly that ASFV pol X undergoes some conformational adjustment in the complex with the dsDNA through the release of anions from the protein. Additional information about the protein conformation flexibility comes from studies of the binding process as a function of temperature (Figure 7(a)–(c)). The simplest thermodynamic model that can account for the observed dependence of the maximum quenching, ΔFmax and the non-linear character of the Arrhenius plot include two conformations of the polymerase, P1 and P2, with

ASFV Pol X-dsDNA Interactions

different intrinsic affinities for the dsDNA. The obtained enthalpy change of the P1 b– P2 transition, ΔHC ∼110 kcal/mol, is large if one considers that ASFV pol X is a relatively small protein of ∼20,000 Da. Although there is a significant error in the value of ΔHC, it suggests strongly that the observed transition includes rearrangements of the protein structure, possibly accompanied by the release of dehydrated ions. Interestingly, the binding of the enzyme to the dsDNA in both conformations, P1 and P2, is temperature-independent. Such temperature-independence indicates that the intrinsic ASFV pol X–dsDNA interactions are, effectively, entropy-driven processes. Sequence-specific protein–nucleic acid interactions were proposed to contain a significant enthalpy contribution to the free energy of binding.61,62 However, the specificity of a DNA repair polymerase such as ASFV pol X relies on DNA structure recognition, not on sequence recognition.61,62 Strong positive cooperative interactions indicate specific orientation of ASFV Pol X in complex with the dsDNA A peculiar aspect of ASFV pol X binding to dsDNA is the presence of significant positive cooperative interactions. Recall, ASFV pol X binds ssDNA without any detectable cooperativity in the binding process.17 The dramatic difference in the nature of enzyme interactions with two different conformations of the nucleic acid indicates very different orientations and/or different conformations of the bound polymerase in both complexes. The specific orientation or conformation of the enzyme must be induced by interactions with the dsDNA. The stiff structure of the dsDNA allows for only limited changes of the nucleic acid conformation in the complex. On the other hand, it also imposes a restricted spatial orientation of the bound enzyme molecules, which must facilitate the engagement of the interacting areas of the protein. Much more flexible ssDNA is less suitable for imposing a restricting orientation of the bound polymerase molecules and rather engages in interactions an additional area of the protein. The changes in the enzyme orientation in complexes with different DNA conformations may play an important role in the damaged DNA recognition (see below). Similar to the intrinsic interactions, the increase of the concentration of salt weakens cooperative interactions. The negative slopes, ∂logω/∂log [NaCl] = −0.6 ± 0.2 and ∂logω/∂log [NaBr] ≈ −1 ± 0.3, show that a net ion release accompanies cooperative interactions. The difference between the net numbers of ion release, observed in the presence of Cl– and Br–, is within experimental accuracy. Nevertheless, it is larger for NaBr than NaCl, indicating that anions participate in the ion-exchange process accompanying cooperative interactions.43,44 Because the DNA does not bind anions, the contribution of anions to the net ion release provides evidence that the cooperative interactions result from direct protein–protein interactions.

91

ASFV Pol X-dsDNA Interactions

Contrary to the early semi-quantitative assessments, the results obtained in this work show clearly that ASFV pol X has a strong intrinsic affinity for dsDNA.15,16 The intrinsic binding constant is of the order of ∼108 M−1, which is approximately two orders of magnitude larger than the intrinsic binding constant for the ssDNA conformation in analogous solution conditions (Table 1).17 This is very surprising if one considers that, in the complex with ssDNA, the enzyme interacts with the DNA, using both DNAbinding subsites of the total DNA-binding site, while in the complex with dsDNA, only the non-catalytic C-terminal domain is engaged in interactions with the nucleic acid. In fact, these data indicate that the DNAbinding subsite on the non-catalytic C-terminal domain is geared specifically for interactions with dsDNA. Moreover, the affinity of the enzyme for dsDNA is additionally amplified by significant positive cooperative interactions, which are absent from the enzyme binding to the ssDNA.17,29 Although the intrinsic affinity and cooperative interactions weaken at higher concentrations of salt, the effect of salt is much less pronounced than in the case of the enzyme binding to the single-stranded conformation of the nucleic acid. Altogether, these results indicate that ASFV pol X will bind predominantly to the dsDNA conformation and exclude the possibility that the enzyme intrinsic affinity for ssDNA plays a significant role in the recognition of the gapped DNA by ASFV pol X. The only other DNA repair polymerase whose interactions with the ssDNA and dsDNA have been addressed quantitatively is the mammalian pol β.18–25,29–31,58,59 The data reported here show that, in spite of very different structures, both enzymes show significant similarity in their interactions with the ssDNA and dsDNA. The total DNA-binding site is structurally and functionally heterogeneous. The proper DNA-binding site is located on the noncatalytic domain. The proper subsite has significantly higher affinity for the nucleic acid than the secondary DNA-binding subsite located on the catalytic domain and shows a significant preference for the dsDNA. Moreover, both enzymes bind the dsDNA with significant positive cooperativity, characterized by a similar cooperativity parameter, ω. Such correspondence of activities between two structurally very different enzymes points to a general functional behavior of the DNA repair polymerases, where the total DNA-binding site is composed of two DNA-binding subsites, which play very different functions in ssDNA gap recognition and catalysis.18–25,29–31,58,59

Materials and Methods Reagents and buffers All chemicals were reagent grade. All solutions were made with distilled, deionized N18 MΩ (Milli-Q Plus) water. Buffer C is 10 mM sodium cacodylate (adjusted to pH 7.0 with HCl), 10% (v/v) glycerol. The temperature

and the concentrations of NaCl, NaBr, and MgCl2 in the buffer are indicated in the text. ASFV Pol X The plasmid harboring the ASFV pol X gene was a generous gift from Dr Maria L. Salas (Universidad Autonoma, Madrid, Spain). The gene for the enzyme was inserted into plasmid pET30a under control of the phage T7 polymerase system. Isolation and purification of the protein was performed essentially as described,6,17 with slight modifications. The concentration of the protein was determined spectrophotometrically using the extinction coefficient ε280 = 1.541 × 104 cm−1 M−1, obtained by an approach based on Edelhoch's method.63,64 Nucleic acids DNA oligomers were purchased from Midland Certified Reagents (Midland, TX). Oligomers were at least N95% pure as judged by PAGE and autoradiography. Labeled ssDNA oligomers contained a fluorescent label, CP or CPM, attached to the 5′ or 3′ end through a six carbon linker. The concentrations of all ssDNA oligomers were determined spectrophotometrically as described.23–25 The dsDNA substrates were obtained by mixing the ssDNA oligomers at given concentrations, warming the mixture for 5 min at 95 °C, and cooling slowly for ∼3–4 h.21–31 The integrity of the dsDNA oligomers was checked by UV melting and analytical ultra-centrifugation techniques.23–25 The melting temperature of the dsDNA oligomers was ∼54 °C or higher under these experimental conditions.21–25,29–31 Analytical ultracentrifugation Analytical ultracentrifugation experiments were performed with an Optima XL-A analytical ultracentrifuge (Beckman Inc., Palo Alto, CA) as described.19,34,64–66 Sedimentation equilibrium scans were collected at the absorption band of CP of the labeled dsDNA oligomers (435 nm) at a large excess of protein (5 × 10−6 M) over DNA (3 × 10−7 M (oligomer)) to ensure complete saturation of the DNA. Non-linear least-squares fits of the scans were performed as described.65 Fluorescence measurements Steady-state fluorescence measurements were performed using the SLM-AMINCO 8100 spectrofluorimeter. In order to avoid artifacts due to the fluorescence anisotropy, polarizers were placed in excitation and emission channels and set at 90° and 55° (magic angle), respectively.17–23,25,32–39,67,68 Formation of the complex was followed by monitoring the fluorescence of the CP-labeled DNA (λex = 435 nm, λem = 480 nm). Computer fits were performed using KaleidaGraph software (Synergy Software, PA) and Mathematica (Wolfram Research, IL). The relative fluorescence quenching of the nucleic acid, ΔF, upon binding the polymerase is defined as ΔF = (F0–Fi)/Fo, where Fi is the fluorescence of the nucleic acid solution at a given titration point i, and F0 is the initial fluorescence of the sample. Time-dependent fluorescence lifetime and anisotropy measurements were done with an IBH 5000 U time-correlated single-photon counting instrument (IBH, Glasgow, UK) equipped with polarizers as well as excitation and emission monochromators as described.68

92

ASFV Pol X-dsDNA Interactions

Determination of the average fluorescence energy transfer efficiency from the donor, the single tryptophan residue of ASFV Pol X, to the acceptor CPM located on the dsDNA The Förster efficiency of the fluorescence energy transfer, E, from the donor, a single tryptophan residue located in the palm domain of ASFV pol X to the acceptor CPM residue, located at the 5′ or 3′ end of a dsDNA oligomer, was determined using two apparent fluorescence energy transfer efficiencies. The energy transfer efficiency, ED, obtained from quenching of the donor fluorescence is defined as:24,29,47,48

 1 FD  FDA ð13Þ ED ¼ FD mD where FD and FDA are the fluorescence of the donor in the absence and in the presence of the acceptor, respectively, νD is the fraction of the donor in the complex with the acceptor. The quantity νD is determined using binding parameters for a given DNA substrate, obtained from the thermodynamic analysis of the enzyme–dsDNA interactions (see above).24,29,47,48 The apparent fluorescence transfer efficiency EA was determined, using the sensitized acceptor fluorescence, by measuring the fluorescence intensity of the acceptor (CPM residue), excited at a wavelength where a donor (tryptophan) predominantly absorbs, in the absence and in the presence of the donor. The fluorescence intensities of the acceptor in the absence, FA, and in the presence, FAD, of the donor are defined as:24,29,47,48 FA ¼ I0 qA CAT fA F

ð14aÞ

FAD ¼ ð1  rA ÞFA þ Io qA rA CAT fA B þ Io qD CDT rD fA B EA

ð14bÞ

and

where Io is the intensity of incident light, CAT and CDT are the total concentrations of the acceptor and the donor, νA is the fraction of acceptors in the complex with donors, εA and εD are the molar absorption coefficients of the acceptor and the donor at the excitation wavelength, respectively, A ϕA F and ϕB are the quantum yields of the free and bound acceptor, and EA is the average transfer efficiency determined by the acceptor sensitized emission. All quantities in equations (14a) and (14b) can be determined experimentally.24,29,47,48 Dividing equation (14a) by equation (14b) and rearranging provides the fluorescence energy transfer efficiency EA as: ! #  
" A !
 1 qA CAT fF FAD fA F þ r þ r  1 EA ¼ A A FA rD qD CDT fA fA B B ð15Þ The Förster energy transfer efficiency E is related to the apparent quantities ED and EA by:65,69 E¼

EA ð1  ED þ EA Þ

ð16Þ

The fluorescence energy transfer efficiency between the donor and the acceptor dipoles E is related to the distance R separating the dipoles by:5 R ¼ Ro

 1 ð1  EÞ 6 E

ð17Þ

where, Ro = 9790(κ2n−4ϕdJ)1/6, is the so-called Förster critical distance (in Å), the distance at which the transfer efficiency is 50%, κ2 is the orientation factor, ϕd is the donor quantum yield in the absence of the acceptor, and n is the refractive index of the medium (n = 1.4), the overlap integral, J, characterizes the resonance between the donor and acceptor dipoles.24,29,47,48,56 Using the spectra of the ASFV pol X and CPM-labeled dsDNA oligomers (data not shown), the Förster critical distance for the tryptophan residue in ASFV Pol X and the acceptor CPM Ro = 21.7 Å.29 The fluorescence transfer efficiency determined for a single donor–acceptor pair depends upon the distance between the donor and the acceptor, R, and the factor κ2 describing the mutual orientation of the donor and the acceptor dipoles. The factor κ2 can assume a value from 0–4. For complete random orientation of the acceptor and the donor, κ2 = 0.67.55,56 On the other hand, because the distance between a donor and an acceptor depends upon the 1/6th power of κ2, only the two extreme values (0 or 4), resulting from a peculiar orientation of the donor and acceptor dipoles and complete dipole immobilization on the time-scale of their fluorescence lifetimes, would affect the determined fluorescence energy transfer efficiencies significantly and, in turn, conclusions about the distances.23,29,47,48 The value of κ2 cannot directly be determined experimentally. However, the effect of κ2 can be assessed experimentally by examining either the multiple donor–acceptor pairs or the same donor–acceptor pair in different orientations and environments.23,29,47,48 Very similar energy transfer efficiencies obtained in such experimental approaches effectively exclude any unfavorable values of κ2. Determination of thermodynamically rigorous binding isotherms of the ASFV Pol X–dsDNA complexes In this work, we followed the binding of ASFV pol X to dsDNA oligomers, by monitoring the fluorescence quenching, ΔF, of the CP-labeled nucleic acid. To obtain quantitative estimates of the total average degree of binding, ∑Θi (average number of bound ASFV pol X molecules per dsDNA oligomer) and the free protein concentration, PF, independent of any assumption about the relationship between the observed spectroscopic signal and ∑Θi, we used an approach described earlier.17–23,25,29,37 Briefly, each different possible i complex of ASFV pol X with dsDNA contributes to the experimentally observed fluorescence quenching, ΔF. Thus, ΔF is functionally related to ∑Θi by: X ð18Þ DF ¼ Qi DFi where ΔFi is the maximum fluorescence quenching of the nucleic acid with ASFV pol X bound in complex i. The same value of ΔF obtained at two different total nucleic acid concentrations MT1 and MT2, indicates the same physical state of the nucleic acid, i.e. the total average degree of binding, ∑Θi, and the concentration of free ASFV pol X, PF, must be the same. The value of ∑Θi and PF is then related to the concentrations of total protein, PT1 and PT2, and the total concentrations of nucleic acid, MT1 and M T2, at the same value of ΔF by: X ðPT2  PT1 Þ ð19Þ Qi ¼ ðMT2  MT1 Þ X Qi MTx ð20Þ PF ¼ PTx  where x = 1 or 2.17–23,25,29,37

ASFV Pol X-dsDNA Interactions Analysis of the ASFV Pol X - unmodified dsDNA complexes using the MCT method Determination of binding parameters for the ASFV pol X - unmodified dsDNA oligomer complex was done using the MCT method, with the dsDNA 10-mer labeled with CP, as a reference fluorescent nucleic acid.25,38 Briefly, if the fluorescent reference nucleic acid at total concentration MTR is titrated with the protein in the absence of a competing unmodified nucleic acid of total concentration, MS, the total concentration of the protein, PT1, at which a given fluorescence change, ΔFi, is observed, is described by the mass conservation relationship: X Qi MTR þ PF ð21Þ PT1 ¼ R

where (∑Θi)R, and PF are the degree of binding of the protein on the reference nucleic acid and the free protein concentration, respectively. The total concentration of the protein, PT2, at which the same ΔFi is observed at the same MTR, in the presence of the competing DNA, is: X Qi MTR þ PF ð22Þ PT2 ¼ S

where (∑Θi)S is the degree of binding of the protein on the non-fluorescent oligomer. Subtracting equation (21) from equation (22) provides equation (23), which allows us to determine the total average degree of binding of the protein on the competing, unmodified dsDNA oligomer, as:25,38 X Qi ¼ ðPT2  PT1 Þ=MS ð23Þ S

Acknowledgements This work was supported by NIH grant GM-58565 to W. B. The authors thank Mrs Gloria Bellard for reading the manuscript.

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Edited by D. E. Draper (Received 23 April 2007; received in revised form 15 June 2007; accepted 18 June 2007) Available online 27 June 2007