# Single Strand DNA Specificity Analysis of Human Nucleoside Diphosphate Kinase B

Fabrice Agou, Sharona Raveh, Sébastien Mesnildrey, Michel Véron

► To cite this version:

Fabrice Agou, Sharona Raveh, Sébastien Mesnildrey, Michel Véron. Single Strand DNA Specificity Analysis of Human Nucleoside Diphosphate Kinase B. Journal of Biological Chemistry, 1999, 274 (28), pp.19630-19638. 10.1074/jbc.274.28.19630. pasteur-03276872

HAL Id: pasteur-03276872

https://pasteur.hal.science/pasteur-03276872

Submitted on 2 Jul 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Single Strand DNA Specificity Analysis of Human Nucleoside Diphosphate Kinase B*

(Received for publication, December 30, 1998, and in revised form, March 22, 1999)

Fabrice Agou, Sharona Raveh, Sébastien Mesnildrey, and Michel Véron‡

From the Unité de Régulation Enzymatique des Activités Cellulaires, Institut Pasteur, CNRS-URA 1773,
25 rue du Docteur Roux 75724, Paris cedex 15, France

Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (≈17 kDa). A distinctive property of human NDPK-B encoded by the gene *nm23-H₂* is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B-DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the *c-myc* oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA-protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of *c-myc* or possibly other genes involved in cellular differentiation.

Nucleoside diphosphate kinases are essential oligomeric enzymes that play a key role in the maintenance of the intracellular pool of all (d)NTPs and NTPs. They catalyze the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate via the formation of an enzymatic intermediate phosphorylated on a catalytic histidine residue (1). The crystal structures of NDP<fnref n="1" /> kinases from several organisms including prokaryotes (2) and eukaryotes (3–5) have been determined. They all share a common subunit fold, built around a βαββαβ motif, also present in the “palm domain” of DNA polymerases (5). Although the prokaryotic enzyme from *Myxococcus xanthus* is a tetramer (6), all eukaryotic enzymes known to date are hexamers composed of identical subunits each containing an active site. The hexameric state is essential for enzymatic

activity, and binding of ADP or ATP contribute to the hexamer stability (7).

In human cells, four members of the NDP kinase family encoded by the genes *nm23-H₁* (8), *nm23-H₂* (9), *DR-nm23* (10), and *nm23-H₄* (11) have been identified so far. A fifth isoform, *nm23-H₅* was isolated very recently but it does not exhibit NDP kinase activity when overexpressed in bacteria (12). Nm23-H₁ and Nm23-H₂ share the highest homology (88%) and can form heterohexamers (13). Nm23-H₂/NDPK-B is present both in the cytoplasm and in the nucleus (14, 15). Phylogenetic analysis (16) and GFP fusion-protein constructs<fnref n="2" /> indicate that Nm23-H₄ is a mitochondrial form of NDP kinase. Altered levels of expression of *nm23-H₁*, *nm23-H₂* and *DR-nm23* are associated with a variety of cellular processes including differentiation, proliferation, apoptosis, and suppression of tumor metastasis (17). Using the serial analysis of gene expression method (18), it was shown that *nm23-H₂* was up-regulated in gastrointestinal tumors (19). Remarkably, the catalytic activity does not explain the role of *nm23* in these cellular processes (17, 20, 21).

A role for the *nm23-H₂* gene product in cell regulation independent from its NDPK activity was first suggested by the observation that this protein binds to nucleic acids. Indeed NDPK-B was identified as a transcription factor based on its ability to stimulate the transcription *in vitro* of the *c-myc* gene (22). This stimulatory activity is independent from the enzymatic activity, because it was retained in a catalytically inactive mutant (23). In addition, transient transfections of a plasmid bearing the –160/–101 upstream nuclease hypersensitive element of the *c-myc* promoter (also termed PuF site or CT element), have shown that NDPK-B increases the expression of a reporter gene by 3–5-fold (24, 25). Moreover, mutations in the PuF site affect the transcriptional efficiency of NDPK-B (25). However Michelotti *et al.* (26) using a one-hybrid system, showed that NDPK-B is not a conventional transcription factor. The nuclease hypersensitive element contains a sequence homopurine/homopolyrimidine-rich, which is prone to form non-B duplex DNA-like intermolecular triplexes (27, 28) or tetraplexes (29). This region is nucleosome-free (30) and forms single strand DNA (31), presumably resulting from the appearance of secondary structure. Using a mobility shift assay, we reported previously a qualitative analysis of the DNA-binding properties of NDPK-B (32), indicating a preference for single strand binding.

In this study, we report the first quantitative analysis of human NDPK-B-DNA complex formation using several biochemical methods. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits strong specificity for single-stranded DNA. Moreover, competition experiments performed with NDPs show that the affinity for DNA is significantly higher than for

<footnote>* This work was supported by grants from the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer and the Agence Nationale de Recherches sur le SIDA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>‡ To whom correspondence should be addressed. Tel.: 33-1-45-68-83-80; Fax: 33-1-45-68-83-99; E-mail: myveron@pasteur.fr.</footnote>

<footnote n="1">¹ The abbreviations used are: NDP, nucleoside diphosphate; DTE, dithioerythreitol; MES, 2-([N-morpholino]ethanesulfonic acid; GFP, green fluorescent protein; PAPS, adenosine 3'-phosphate 5'-phosphosulfate.</footnote>

<footnote n="2">² M. Lacombe, personal communication.</footnote>
<caption>TABLE II
Purification of recombinant NDPK-B</caption>

All purification steps were performed at 4 °C. One unit of activity corresponds to the amount of dTDP (micromoles) transformed/min at 25 °C.

<table><thead><tr><th>Purification step</th><th>Total protein</th><th>Total activity</th><th>Specific activity</th><th>Yield</th><th>Relative purification factor</th></tr><tr><td></td><td>mg</td><td>units</td><td>units/mg</td><td>%</td><td></td></tr></thead><tbody><tr><td>Crude extract</td><td>2,855</td><td>195,500</td><td>69</td><td>100</td><td>1</td></tr><tr><td>Polymin-P supernatant</td><td>1409</td><td>182,250</td><td>130</td><td>93</td><td>2</td></tr><tr><td>POROS HQ</td><td>487</td><td>169,650</td><td>348</td><td>87</td><td>5</td></tr><tr><td>POROS HS</td><td>90</td><td>118,215</td><td>1,314</td><td>61</td><td>19</td></tr><tr><td>Ceramic hydroxyapatite</td><td>60</td><td>84,000</td><td>1,400</td><td>43</td><td>20</td></tr></tbody></table>

where $[DNA]_T$, $[P]_T$ are the total concentration of the radiolabeled oligonucleotide and NDPK-B, respectively, and $I$ is the free concentration of inhibitor NDP, which in our conditions is very close to the total concentration. $K_D$ is the dissociation constant for the DNA-NDPK-B interaction and $K_I$ is the inhibition constant for the NDP-NDPK-B interaction.

**Fluorescence Studies**—All fluorescence measurements were performed on a PTI spectrofluorometer Quantamaster™ at 20 °C, in a buffer 20 mM potassium phosphate, pH 7.0, containing 75 mM potassium chloride, 5% glycerol, 5 mM magnesium chloride, and 1 mM DTE. The excitation wavelength was 304 nm to minimize the contribution of tyrosyl residues to the total fluorescence and the inner filter due to light absorption by the oligonucleotide. Protein fluorescence was measured at 340 nm and the excitation and emission bandwidths were both set to 2.5 nm. The oligonucleotide titrations were carried out with NDPK-B monomeric concentrations of 0.5–2 µM (1 ml) by adding varying amounts of DNA to a constant concentration of NDPK-B. In all titration experiments, the progressive dilution during the course of titration never exceeded 5%. Following the addition of the saturating oligonucleotide concentration, the equilibrium state was checked by recording the protein fluorescence during 5 min. In the fluorescence titrations each reading was collected for 60 s after a 1-min period of equilibration. The extent of photobleaching was determined by performing experiments without adding the oligonucleotide. The decrease in fluorescence due to photobleaching is less than 0.6% in a 15-point titration.

**Calculation of Binding Parameters**—Bimolecular binding data were fitted to the Langmuir binding equation. The independent variable in these experiments was either total oligonucleotide or total NDPK-B concentrations. The equation was then converted to a function of total NDPK-B and total oligonucleotide for fluorescence experiments (Eq. 5);

$$[DNA \cdot P] = K_D + [DNA]_T + n[P]_T - \sqrt{K_D + [DNA]_T + n[P]_T - 4[DNA]_T n[P]_T} = A \quad (Eq. 5)$$

where $K_D$ represents the dissociation constant, $[P]_T$ is the total NDPK-B concentration, $n$ is the number of equivalent binding sites, and $[DNA]_T$ is the total oligonucleotide concentration (a similar scheme can be worked out for titration of DNA by NDPK-B using the filter-binding assay; $[DNA]_T$ is substituted for $n[DNA]_T$ and $n[P]_T$ for $[P]_T$). For fluorescence experiments, because the quenching was directly proportional to binding, the theoretical quenching was $F_{obs} = (F_0 - F_{max})A' + F_0$ (Eq. 5') where $F_0$ is the initial fluorescence intensity before addition of the oligonucleotide, $F_{max}$ is the fluorescence after saturation with the oligonucleotide, and $A'$ is the molar fraction of protein site occupied. In the filter-binding assay, the fraction of oligonucleotide retained on a filter, $R$, was calculated as follows: $(C - C_B)/(C_T - C_B)$ where $C$ is the counts retained on the filter, $C_B$ is the counts obtained in the absence of NDPK-B, and $C_T$ is the total input counts. Experimental data were fitted according to the symmetric equation derived from Equation 5, including the filter efficiency, $E$, as follows: $R = E \cdot A''$ where $A''$ is the molar fraction of oligonucleotide bound. All experimental data were fitted by nonlinear regression analysis using the Levenberg-Marquardt algorithm in the commercially available graphics software package KaleidaGraph (Synergy Software, Reading PA). For fluorescence experiments, because it is hazardous to fit the experimental data with two parameters estimated ($n$ and $K_D$), we determined graphically the stoichiometry $n$ using a NDPK-B concentration (2 µM) higher than the $K_D$ expected, such that the abscissa of the intersection between the initial tangent and the asymptote is practically equal to $n \cdot [NDPK-B]_T$. Then, the $K_D$ was calculated by fitting the equation (Eq. 5') using a NDPK-B concentration of 0.5 µM.

**Analytical Ultracentrifugation**—Sedimentation equilibrium experiments were performed at 9,000, 12,000, 16,000, and 18,000 rpm at 15 or 10 °C in a Beckman Optima XL-A analytical ultracentrifuge, using an

An-60 Ti rotor and a double-sector cell of 12-mm path-length. Enzyme samples (400 µl) were dialyzed exhaustively against three changes of buffer (500 ml each) described in Table III. Equilibrium was verified from the superimposition of duplicate scans recorded at 3 h intervals. The experimental data were first fitted to a model for a single homogeneous species following Equation 6 (38),

$$c_{(r)} = c_{(ref)} \exp\left[\left[\left(M(1 - \bar{\nu}\rho)\omega^2/2RT\right)(r^2 - r_{ref}^2)\right] + \delta\right] \quad (Eq. 6)$$

where $c_{(r)}$ is the concentration of the protein at radial position $r$, $c_{(ref)}$ is the concentration of the protein at an arbitrary reference distance $r_{ref}$, $M$ is the molecular mass, $\bar{\nu}$ is the partial specific volume of the solute, $\rho$ is the density of the solvent, $\omega$ is the angular velocity, $R$ and $T$ are the molar gas constant and the absolute temperature, respectively, and $\delta$ is the base-line offset. The partial specific volume was calculated from the amino acid composition of the protein, and the density of the buffers was determined from published tables (38). After collecting data at sedimentation equilibrium, samples were centrifuged at 40,000 rpm for 12 h to sediment the protein, and radial scans were again collected to obtain a base-line correction for each cell. In all cases, because inclusion of a second virial coefficient gave no improvement of the fit, nonideality in the system was not detected.

## RESULTS

**Purification of Recombinant NDPK-B**—Because human NDPK-B is a basic protein, its ability to bind nucleic acids in cellular extracts could interfere with the purification process. Indeed, the protein obtained using the previous purification procedure (32, 39) was contaminated by nucleic acids. We developed a new purification method including a precipitation of the nucleic acids with Polymin-P, followed by three ion-exchange chromatography steps (see Table II). It should be noted that, despite Polymin-P precipitation and the passage on a strong anion-exchanger (POROS HQ), nucleic acids were still present in the flow-through fraction containing NDP kinase activity, as shown by the absorption spectrum of the protein. Two additional steps on cation-exchanger columns with extensive washing, were necessary to obtain a pure protein free from nucleic acids as judged from SDS-polyacrylamide gel electrophoresis stained by silver nitrate and from the shape of the absorption spectra (data not shown).

**Oligomeric State of Recombinant NDPK-B**—A tetrameric structure was recently reported for human recombinant NDPK-B expressed in *E. coli* (39). However, the crystal structure of NDPK-B (5, 40) showed that its oligomeric state is $\alpha_6$. We previously demonstrated that DNA binding to the Dictyostelium enzyme is strongly dependent on its oligomeric state. Indeed, while a dimeric mutant from Dictyostelium binds to a single strand oligonucleotide, the hexameric wild-type does not (41). We checked the oligomeric state of our purified recombinant NDPK-B by equilibrium sedimentation. Fig. 1 shows the experimental distribution at 12,000 rpm under buffer conditions used by Schaertl (39). The relative concentration of NDPK-B was fitted to an ideal monodisperse solute of 105,000 ± 3,000 (Table III). Considering that the initiator methionine is removed by methionine aminopeptidase (39, 42), this value precisely matches the theoretical calculated mass of the hexameric species 103,002 Da (6 × 17, 167 Da). The distribution of NDPK-B, obtained at two other rotor speeds (9,000
<figure><img src="image_2.png" /><figcaption>TABLE III
Sedimentation equilibrium analysis of human NDPK-B</figcaption></figure>

**FIG. 1.** Sedimentation equilibrium analysis of NDPK-B. Relative concentration of NDPK-B measured by absorbance at 280 nm is plotted as a function of radial distance after sedimentation under the conditions described in Table III at 12,000 rpm. Experimental values (○) were fitted (curve) as described under "Experimental Procedures." The line shows the best-fitting curve for an ideal monospecies with a molecular mass equivalent to 105,000 Da. The distribution of residuals is shown above.

Experimental conditions were similar to those used by (39). Molecular masses were obtained by fitting Equation 6 described under "Experimental Procedures" in a single ideal solute model. The enzyme behaved as a monodispersers entity in each of the runs.

<table><thead><tr><th>Buffer</th><th>Temperature</th><th>Initial protein concentration</th><th>Speed</th><th>Molecular mass observed</th><th>Molecular species</th></tr><tr><td></td><td>°C</td><td>mg/ml</td><td>rpm</td><td>Da</td><td>%</td></tr></thead><tbody><tr><td>50 mM Tris-HCl, pH 8</td><td></td><td></td><td>9,000</td><td>97,000 ± 8,000</td><td>100 α<sub>6</sub></td></tr><tr><td>5 mM MgCl<sub>2</sub></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1 mM EDTA</td><td>15</td><td>0.2</td><td>12,000</td><td>105,000 ± 3,000<sup>a</sup></td><td>100 α<sub>6</sub></td></tr><tr><td>100 mM KCl</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1 mM DTE</td><td></td><td></td><td>18,000</td><td>100,000 ± 4,000</td><td>100 α<sub>6</sub></td></tr><tr><td>10 mM MES, pH 6.0</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>5 mM MgCl<sub>2</sub></td><td>10</td><td>0.2</td><td>16,000</td><td>106,000 ± 6,000</td><td>100 α<sub>6</sub></td></tr><tr><td>1 mM DTE</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

and 18,000 rpm) could also be fitted to a single ideal homogeneous species with a molecular mass corresponding to a hexamer (Table III), indicating that no dissociated form could be detected in our enzymatic preparation. The stability of the hexameric protein was also examined. Lowering the temperature to 10 °C and removing KCl in a more acidic buffer, pH 6, did not alter the oligomeric structure (Table III).

Characterization of the NDPK-B·Single Strand DNA Complex—We first quantified the interactions between NDPK-B and DNA by the filter-binding assay, a fast and simple method. We used the purine-rich single strand of the *c-myc* promoter, because it has been shown previously that most of purine bases composing this strand are protected *in vitro* by the complex DNA·NDPK-B after treatment with chemical probe (*c-myc* / ssPu-29, Table I) (25, 43). Fig. 2 shows a typical binding isotherm for the interaction of various concentrations of protein with a fixed concentration of either the *c-myc* / ssPu-29 oligonucleotide or another purine-rich strand, the AML / ssPu-30 oligonucleotide from the major late promoter of adenovirus (Table I). In each case, the shape of the binding curve is not sigmoidal, and the corresponding Scatchard plot is linear (Fig. 2, inset), indicating that NDPK-B binds to the oligonucleotide without cooperativity. Assuming a filter-retention efficiency of 85 and 80% for *c-myc* and AML oligonucleotides, respectively, the stoichiometry of the two complexes computed from the intersection point of the two linear portions of the curve corresponds to one oligonucleotide bound per protein subunit. Taking into account this stoichiometry, the apparent *K*<sub>D</sub> for *c-myc* / ssPu-29 and AML / ssPu-30 is 140 nM and 260 nM, respectively, indicating a weak preference for the *c-myc* strand as compared with the AML strand.

pH and Salt Dependence of the Binding of NDPK-B to DNA— The stability of the *c-myc*·ssPu complex with NDPK-B under several pH is shown in Table IV. The dissociation constant is 17-fold higher at pH 8.5 than at pH 5.5, indicating that at acidic pH the stability of the nucleoprotein complex is increased. At pH 5.5 or 7 and with a washing buffer at pH 5.5 (Table IV), similar *K*<sub>D</sub> values (140 and 100 nM, respectively) were measured. This shows that the stability of complex retained on the filter depends essentially on the acidic pH of the washing buffer under reduced pressure. This underlines the lability of NDPK-B-single strand DNA interactions.

The KCl concentration dependence of the dissociation constant for the *c-myc* / ssPu-29 oligonucleotide was also examined (Table V). Increasing the KCl concentration from 20 to 200 mM reduces the apparent affinity by a factor of more than 50, revealing a strong contribution of the polyelectrolyte effect to the stability of the complex under our titration conditions.

<footnote><sup>a</sup> The sedimentation profile of human NDPK-B is shown in Fig. 1.</footnote>
FIG. 2. Determination by the filter-binding assay of the dissociation constant of c-myc/ssPu-29:NDPK-B complex. The oligonucleotide ³²P-c-myc/ssPu-29 (83 nM) or ³²P-AML/ssPu-30 (52 nM) was incubated with the indicated concentrations of NDPK-B under conditions described under "Experimental Procedures." Solid curves represent the nonlinear least-square fits using the Langmuir binding equation. Inset: Scatchard plot (n/L versus n) of the binding of c-myc/ssPu-30 showing a linear plot (noncooperative effect) and the stoichiometry of the oligonucleotide binding (intercept on the abscissa). Protein concentration corresponds to monomers of NDPK-B.

<figure><img src="image_3.png" /><figcaption>TABLE IV
pH dependence of NDPK-B binding to c-myc/ssPu-30</figcaption></figure>

All assays were performed at 4 °C. Binding buffer, 20 mM Tris/acetic acid/MES containing 10 mM MgCl₂, 20% glycerol, and 1 mM DTE; washing buffer, same as binding buffer except for the additional presence of 20 mM KCl. The dissociation constants were measured by the filter-binding assay as described under "Experimental Procedures."

<table><caption>TABLE V
Dependence of NDPK-B binding to c-myc/ssPu-30 on KCl concentration</caption><thead><tr><th>Binding buffer</th><th>Washing buffer</th><th>K<sub>D</sub></th></tr><tr><th>pH</th><th></th><th>nM</th></tr></thead><tbody><tr><td>5.5</td><td>5.5</td><td>140 ± 30</td></tr><tr><td>6</td><td>6</td><td>330 ± 60</td></tr><tr><td>7</td><td>5.5</td><td>100 ± 10</td></tr><tr><td>7</td><td>7</td><td>1,600 ± 300</td></tr><tr><td>8</td><td>8</td><td>2,400 ± 400</td></tr></tbody></table>

All experiments were done at pH 5.5 and 4 °C in 20 mM Tris/acetic acid/MES buffer containing 10 mM MgCl₂, 20% glycerol, and 1 mM DTE. The dissociation constants were measured by the filter-binding assay as described under "Experimental Procedures."

<table><thead><tr><th>[KCl]</th><th>K<sub>D</sub></th></tr><tr><th>mM</th><th>nM</th></tr></thead><tbody><tr><td>20</td><td>140 ± 30</td></tr><tr><td>50</td><td>350 ± 50</td></tr><tr><td>100</td><td>2,600 ± 700</td></tr><tr><td>200</td><td>6,500<sup>a</sup> ± 1,000</td></tr></tbody></table>

<sup>a</sup> This value was estimated in a protein concentration range from 1 to 25 µM.

When the data of Table V were plotted as $-\ln[K_D]$ versus $\ln[K_{Cl}]$, a linear dependence of the binding on KCl concentration was found (Fig. 3). The slope of the linear regression of the data allows the calculation of the number of counterions ($K^+$) released from backbone phosphates upon binding of the protein (36). A value of $n = 1.73$ was obtained. Because $\Psi$, the fraction of a counterion bound per phosphate for single strand DNA is 0.71 (36), $m'$ corresponding to the number of ion pairs formed between basic residues of monomeric NDPK-B and the phosphate backbone is equal to 2.4. The non-electrostatic contributions to the free energy of binding can also be estimated using Equation 3 (see "Experimental Procedures") (44). In our case, the value of $\Delta G^0_{obs}$ is about $-4.0 \text{ kcal} \cdot \text{mol}^{-1}$ (extrapolated from the plot of $\ln[K_D]$ versus $\ln[K^+]$ at 1 M). Assuming that $Na^+$ and

<figure><img src="image_4.png" /><figcaption>FIG. 3. Dependence of the binding of NDPK-B to c-myc/ssPu-30 on the KCl concentration. The oligonucleotide (80 nM) interacted with NDPK-B in buffer (20 mM Tris/Acetate/MES (pH 5.5), 10 mM MgCl₂, 20% glycerol, and 1 mM DTE) containing potassium chloride 20, 50, 100, and 200 mM. The data were analyzed as described under "Experimental Procedures." $m'$ is the estimated number of ion pairs formed in the complex.</figcaption></figure>

$K^+$ have an equivalent effect, that $\Delta G^0_{Lys} = \Delta G^0_{Arg} = 0.18 \text{ kcal} \cdot \text{mol}^{-1}$, and that $N$ represents the ions released (36), the non-electrostatic contributions $\Delta G^0_{ne}$ corresponds to $-4.4 \text{ kcal} \cdot \text{mol}^{-1}$. This indicates that under our filter-binding conditions about 50% of the total free energy is contributed by electrostatic interactions.

Competition by NDPs of the Binding of c-myc/ssPu-29 to NDPK-B—In an attempt to determine whether DNA binding involves the mono-NDP-binding site of the protein, we performed competition assays of oligonucleotide binding to NDPK-B in the presence of GDP, ADP, dTDP, or CDP. To improve the sensitivity of the competition assay, we used variable concentrations of NDP and a constant concentration of radiolabeled oligonucleotide and protein such that [NDPK-B] $\approx$ [oligonucleotide] $\approx K_D$. As shown in Fig. 4, each of the four NDPs competed with NDPK-B binding to c-myc/ssPu-29. A representative fit using a quadratic equation (see Eq. 4 under
<figure><img src="image_5.png" /><figcaption>FIG. 4. Competition by NDPs of the binding of c-myc/ssPu-30 to NDPK-B. NDPK-B (120 nM, 30 µl) was incubated for 30 min at 4 °C with radiolabeled c-myc/ssPu-30 (70 nM) in the presence of variable concentrations of GDP (■), ADP (○), dTDP (●), or CDP (□) under the conditions described under "Experimental Procedures."</figcaption></figure>

"Experimental Procedures") yielded $K_I$ values of 17, 70, 80, and 900 µM for GDP, ADP, dTDP, and CDP, respectively. The inhibitory effect of CDP is 50-fold lower than that of GDP, demonstrating a base specificity of the inhibition. It is interesting to note that a similar base specificity has also recently been reported for the enzymatic activity of the human NDPK-B (45) and of the Dictyostelium enzyme (46). Moreover, analysis of the inhibition curve in the presence of GTP gives a $K_I$ equal to 40 µM similar to that of GDP, confirming that formation of the phosphohistidine intermediate does not affect the binding of the oligonucleotide. Taken together, these results suggest strongly that the mononucleotide-binding site acts as binding template for interaction with the oligonucleotide. In addition, we show that the affinity for the best substrate, GDP, determined by stopped-flow experiments (45) is about 150-fold less than for the c-myc/ssPu-29.

*Measurement of Affinities in Solution*—To further characterize the interaction between NDPK-B and the upstream element of the c-myc gene, we used the filter-binding assay and fluorescence spectroscopy to determine whether NDPK-B binds to DNA with sequence specificity. Several oligonucleotides varying in sequence, structure, and length, both from the top and the bottom strands of the c-myc promoter, were used (Table I). Under the standard conditions described above, no complex was isolated by the filter-binding assay with pyrimidine-rich strands like c-myc/ssPy-30 or AML/Py-30. Similarly, only 15% of the pyrimidine strand (CT)₉ or 18% of (CT)₂₇ were retained on the filter at a saturating concentration of NDPK-B (data not shown). Because the electrostatic interactions contribute 50% of the total free energy in our filter-binding conditions as shown above, we concluded that this lability of the complex with the pyrimidine strand was probably due to the protonation of cytosine at pH 5.5. It thus appeared that the filter-binding assay was not appropriate to monitor the sequence specificity of the binding.

To measure affinities in solution, we used the fluorescence titration method to monitor the binding of oligonucleotides to NDPK-B. The measure of intrinsic protein fluorescence is the most accurate method to determine the stoichiometry of oligonucleotide binding because, contrary to the filter-binding assay, each fluorescence titration was performed using constant protein and variable oligonucleotide concentrations. Fig. 5

shows a typical emission spectrum where the excitation wavelength was 304 nm to minimize the absorptive screening effects (inner filter). The presence of a saturating concentration of c-myc/ssPu-29 results in a weak (5%) yet reproducible quenching of the intrinsic fluorescence of NDPK-B with no shift in the emission maximum at 348 nm. This fluorescence quenching by the oligonucleotide could be used to study the binding of various other oligonucleotides to NDPK-B.

To determine the relationship between the extent of fluorescence quenching and the fraction of protein bound, a series of fluorescence titrations with the c-myc/ssPy-30 pyrimidine-rich strand or c-myc/ssPu-29 purine-rich strand was carried out at pH 5.5 or pH 7.0 (Fig. 6 and Table VI). For these studies, we used a higher salt concentration (75 mM potassium chloride and 20 mM potassium phosphate) and a higher temperature (20 °C) to reduce the polyelectrolyte effect as described above, and to increase the hydrophobic contribution to the total free energy. Fig. 6 shows several fluorescence titrations of 30-mer oligonucleotides with NDPK-B at pH 5.5 or pH 7.0. Except with c-myc/ssPy-30 at pH 5.5 where the $K_D$ is high, the stoichiometry computed from the intercept between the tangent of the initial part of the $\Delta F$ and the asymptote is equal to 1 oligonucleotide bound per 2.5 monomer. Taking this stoichiometry into account, a $K_D$ of 35 nM was obtained at pH 5.5 for the c-myc/ssPu-29 strand. In contrast, the affinity of c-myc/ssPy-29 was very poor ($K_D$ > 5 µM). This result confirms the previous filter-binding data, and indicates an excellent correlation between the fluorescence quenching and the fraction of NDPK-B bound to the oligonucleotide. No significant difference in the $K_D$ of the c-myc/ssPu-29 complex was observed at pH 5.5 or pH 7, showing that binding of DNA by NDPK-B is not affected under acidic conditions. Interestingly, a $K_D$ of 83 nM was measured at pH 7.0 with the c-myc/ssPy-30 pyrimidine strand. This affinity is very similar to that of the purine strand c-myc/ssPu29 (45 nM), indicating only a weak preference for the 29-mer c-myc purine-rich strand. Similar results with about 2-fold difference were obtained with shorter oligonucleotides (10-mer) purine-rich or pyrimidine-rich strands corresponding to only one repeat of the motif contained in the positive cis-element of the c-myc gene (see Table I). Thus, lowering the length of the oligonucleotide from 30- to 10-mer did not change the stoichiometry and affinity, suggesting that the main oligonucleotide/protein contacts are preserved within the 10-mer oligonucleotides. The fact that NDPK-B binds to these oligonucleotides with high but similar affinity shows that its recognition of DNA is not sequence specific. These data are further supported by the fact that another promoter (AML) displays a high affinity ($K_D$ = 70 nM) for NDPK-B (Table VI). Why then does it recognize specially the upstream positive element of c-myc gene? To clarify this point, we measured the dissociation constant of the c-myc 30-mer in its double-stranded form (c-myc/ds-30). In contrast with single strand forms c-myc/ssPu-29 or c-myc/ssPy-30, the addition of c-myc/ds-30 to a constant protein concentration during fluorescence titration, generates a quenched signal which diminishes slowly as a function of time (0.2%/min), reflecting that the fluorescence titration with the double-stranded oligonucleotide is not at equilibrium. This is explained by the fact that single strand is formed from double strand in the presence of NDPK-B, as previously demonstrated using a single-strand generation assay (32). Assuming that the c-myc/ds-30 binding occurs very quickly under our conditions, we could estimate an apparent $K_D$ of 5 µM for the double strand oligonucleotide, corresponding to an affinity for c-myc/ds-30 about 110-fold less than for the purine-rich single strand, c-myc/ssPu-29.

Taken together, these results suggest that specific recogni-
FIG. 5. Quenching of the fluorescence emission spectrum of NDPK-B by the c-myc/ssPu-29 oligonucleotide. The intrinsic fluorescence emission spectra of NDPK-B (1 µM) were recorded at 20 °C in the presence or absence of c-myc/ssPu-29 (2 µM) in 20 mM potassium phosphate, pH 7.0, containing 75 mM potassium chloride, 5% glycerol, 5 mM MgCl₂, and 1 mM DTE. The excitation wavelength was 304 nm.

<figure><img src="image_6.png" /><figcaption>TABLE VI
Dissociation constants measured by fluorescence titration</figcaption></figure>

The intrinsic fluorescence of NDPK-B was measured in the presence of several oligonucleotides in 20 mM potassium phosphate, pH 7.0, buffer or 20 mM Tris/acetic acid/MES, pH 5.5, as indicated, both containing 75 mM potassium chloride, 5% glycerol, 5 mM MgCl₂, and 0.5 mM DTE. Fluorescence excitation and emission wavelengths were 304 and 340 nm, respectively. Stoichiometry values presented here result from the analysis of two independent saturation curves, similar to those shown in Fig. 6, performed with 0.5 and 2 µM of NDPK-B. The stoichiometry is calculated for monomers of NDPK-B.

<table><thead><tr><th rowspan="3">Oligonucleotide</th><th colspan="2">K<sub>D</sub></th><th rowspan="3">Stoichiometry</th></tr><tr><th>pH 5.5</th><th>pH 7</th></tr><tr><th colspan="2">nM</th></tr></thead><tbody><tr><td>c-myc/ssPy-30</td><td>2,000 ± 200</td><td>85 ± 10</td><td>0.4</td></tr><tr><td>c-myc/ssPu-29</td><td>35 ± 7</td><td>45 ± 7</td><td>0.4</td></tr><tr><td>c-myc/ds-30</td><td>nd</td><td>5,000 ± 400</td><td>0.4</td></tr><tr><td>c-myc/ssPu-10</td><td>nd</td><td>30 ± 5</td><td>0.4</td></tr><tr><td>c-myc/ssPy-10</td><td>nd</td><td>80 ± 8</td><td>0.4</td></tr><tr><td>AML/ssPu-30</td><td>nd</td><td>70 ± 10</td><td>0.4</td></tr></tbody></table>

<figure><img src="image_7.png" /></figure>

FIG. 6. Fluorescence titration of NDPK-B by c-myc purine-rich or pyrimidine-rich strands. Fluorescence measurements were carried out as described under “Experimental Procedures,” using an excitation wavelength of 304 nm and recording the emission at 340 nm. Samples contained 20 mM KPO₄, pH 7.0 (○, □) or 20 mM Tris/acetic acid/MES, pH 5.5 (●, ■), 75 mM KCl, 5 mM MgCl₂, 5% glycerol, 1 mM DTE, 500 nM monomeric NDPK-B, and varying amounts of c-myc/ssPu-29 (○, ●) or c-myc/ssPy-30 (□, ■). The lines are the best-fit curves using Eq. 5 described under “Experimental Procedures.”

tion of the upstream element of c-myc is essentially due to the presence of a single strand form in this DNA region rather than to base-specific contacts with the particular sequence or to a predominant homopurine/homopyrimidine composition of the stretch of DNA involved in the interaction.

DISCUSSION

In this study, two independent quantitative methods were employed to characterize the DNA-binding properties of human NDPK-B, the product of gene *nm23-H2*. We found that NDPK-B binds to oligonucleotides with considerably higher

affinity as compared to its enzymatic substrates. However, different stoichiometries of the complex were observed, depending on the method and the type of titration used. In the filter-binding assay, the oligonucleotide concentration used was near the dissociation constant, conditions in which the stoichiometry could be in error (47). In contrast, a fixed protein concentration and variable oligonucleotide concentrations, such that [protein] ≫ K<sub>D</sub>, were used for fluorescence titration. Hence, the stoichiometry of 0.4 oligonucleotide per protein subunit derived from the fluorescence experiment is much more accurate. This corresponds to an average of 2.4 oligonucleotides bound per hexamer, assuming that the enzyme does not dissociate upon binding oligonucleotides. If the oligonucleotide binding involves the six mononucleotide binding sites as suggested by competition experiments, these results may indicate that the binding of one oligonucleotide prevents the fixation of a second strand to an adjacent protein site. It is interesting to note that a different stoichiometry (of 1:1) was observed when NDPK-B intrinsic fluorescence was titrated by dinucleotides dAdG or dGdA (data not shown), indicating that the fluorescence signal is linearly dependent on the number of bound ligands. Schnei-
der et al. (46) reported recently that the nucleotide analogue PAPS can adopt a new conformation inside the active site of Dictyostelium NDP kinase. Indeed, the 5'-phosphate of bound PAPS points out from the binding pocket, whereas the 3'-phosphate binds near the active site histidine. It is tempting to speculate that this new binding mode may also apply to the binding of an oligonucleotide to NDPK-B.

The potassium chloride dependence of NDPK-B-binding to c-myc/ssPu-29 shows that 2.4 ion pairs between basic residues of each monomer of NDPK-B and the phosphate backbone of the oligonucleotide contribute to the formation of the NDPK-B-DNA complex. These results are in accordance with those of Postel et al. (48) who showed that two strictly conserved basic residues (Arg-34 and Lys-135) are major determinants of the DNA binding.

In a previous study (32) using the mobility shift assays, we showed that NDPK-B binds preferentially single-stranded pyrimidine-rich sequences. However, this assay could be biased because of the possibility that a double strand could be generated with purine-rich (but not with pyrimidine-rich) sequences in the presence of an excess of poly(dI-dC). As shown above, the affinity for single-stranded DNA is more than 100-fold better than for a double strand, which is likely to explain why no band-shift was observed with the c-myc/ssPurine strand in the presence of an excess of poly(dI-dC). In this study, filter binding failed to demonstrate sequence specificity, although the acidic conditions used are appropriate to isolate protein-oligonucleotide complexes. However, such specificity is clearly demonstrated using the fluorescence method. NDPK-B binds with high affinity either poly-purine or poly-pyrimidine strands, albeit with a slight preference for the c-myc/purine-rich strand. This indicates that the enzyme recognizes DNA without significant sequence specificity. However, the finding that NDPK-B discriminates single strand from double strand oligonucleotides is important, and it may explain how human NDPK-B recognizes specifically the –100 to –150 upstream element of the P1 promoter (49). Indeed this element forms single-stranded DNA, according to the mapping with chemical (like KMnO₄) or enzymatic (like S1 nuclease) single strand probes. Other single strand DNA-binding proteins that bind to this upstream element and act on the regulation of c-myc expression have also been reported (50). These include the ribonucleoprotein hRNP-K, a single strand-specific transcription factor that interacts with at least two nonadjacent repeats of a cytidine-rich nonanucleotide. hRNP-K forms protein-protein interactions with components of the RNA polymerase II transcription machinery such as the TATA-binding protein (31), and its efficiency for c-myc gene activation is strongly dependent on the presence of single strand DNA (51). Interestingly, the specificity of NDPK-B for single strand oligonucleotides compared with double strands shown in this paper is even more pronounced than that reported for hnRNP-K (31). We propose that the transcriptional activation of c-myc by NDPK-B could be due to its ability to stabilize or to generate the cytosine-rich element in a single strand form to allow hnRNP-K to recognize its DNA site more efficiently. This mode of action could be related to the hexameric structure of NDPK-B containing six putative binding sites to anchor single-stranded DNA. This mechanism might be enhanced in a dimeric nuclear NDPK-B, which could be generated in tumoral cells because of an alteration of the hexameric interface. Indeed, a dimeric NDP kinase from Dictyostelium generated by site-directed mutagenesis has an affinity for single strand DNA 500-fold higher than for the hexameric form (41). Whereas these results were obtained by the filter-binding assay, similar evidence was also obtained using

fluorescence titration.<fnref n="3" /> Assuming a similar behavior for human NDPK-B, an affinity for single strand equal to about 0.5 nM could be predicted and thereby would accentuate the transcriptional activation of c-myc.

In a more general way, the single strand specificity of DNA binding by NDPK-B demonstrated in this study could be a clue to explaining a more general role of this protein in transcription regulation. Indeed, NDPK-B could generate a single-stranded hinge in DNA that would allow the capacity of many cis elements to cooperate even when these elements are distant from each other. The relative abundance of NDPK-B presumably required for maintenance of the nucleoside triphosphate pool could then promote or restrict the interaction of conventional transcription factors during differentiation. An effect of NDPK-B on single-stranded DNA flexibility might also contribute to mitosis-specific single-stranded features at the promoters of active genes (52). Finally, this single-stranded specificity predicts another physiological role involved in the maintenance of discrete segments in the unwound state, which may associate the enzyme to other cellular process such as DNA recombination and replication.

**Acknowledgments**—We thank Dominique Deville-Bonne for expert advice in fluorescence spectroscopy, Professor Joël Janin for continuous support, and Dr. Jean-Pierre Waller for critical review of this manuscript. We are indebted to G. Batelier for performing the analytical ultracentrifuge experiments. The excellent technical assistance of Manuel Babolat and the help of Yuxing Sarah Fan during part of this work are gratefully acknowledged.

REFERENCES

1. Agarwal, R. P., Robison, B., and Parks, R. E. J. (1978) *Methods Enzymol.* **51**, 376-385

2. Williams, R. L., Oren, D. A., Munoz-Dorado, J., Inouye, S., Inouye, M., and Arnold, E. (1993) *J. Mol. Biol.* **234**, 1230-1247

3. Dumas, C., Lascu, I., Morera, S., Glaser, P., Fourme, R., Wallet, V., Lacombe, M.-L., Veron, M., and Janin, J. (1992) *EMBO J.* **11**, 3203-3208

4. Chiadmi, M., Moréra, S., Lascu, I., Dumas, C., LeBras, G., Veron, M., and Janin, J. (1993) *Structure* **1**, 283-293

5. Morera, S., Lacombe, M.-L., Xu, Y., LeBras, G., and Janin, J. (1995) *Structure* **3**, 1307-131453

6. Williams, R. L., Oren, D. A., Munoz-Dorado, J., Inouye, S., Inouye, M., and Arnold, E. (1993) *J. Mol. Biol.* **234**, 1230-1247

7. Mesnildrey, S., Agou, F., Karlsson, A., Bonne, D. D., and Veron, M. (1998) *J. Biol. Chem.* **273**, 4436-4442

8. Steeg, P. S., Bevilacqua, G., Kopper, L., Thorgeirsson, U. P., Talmadge, J. E., Liotta, L. A., and Sobel, M. E. (1988) *J. Natl. Cancer Inst.* **80**, 200-204

9. Stahl, J. A., Leone, A., Rosengard, A. M., Porter, L., King, C. R., and Steeg, P. S. (1991) *Cancer Res.* **51**, 445-449

10. Venturelli, D., Martinez, R., Melotti, P., Casella, I., Peschle, C., Cucco, C., Spampinato, G., Darzynkiewicz, Z., and Calabretta, B. (1995) *Proc. Natl. Acad. Sci. U. S. A.* **92**, 7435-7439

11. Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. (1997) *Hum. Genet.* **99**, 550-557

12. Munier, A., Feral, C., Milon, L., Phung-Ba Pinon, V., Gyapay, G., Capeau, J., Guellaen, G., and Lacombe, M. L. (1998) *FEBS Lett.* **434**, 289-294

13. Gilles, A. M., Presecan, E., Vonica, A., and Lascu, I. (1991) *J. Biol. Chem.* **266**, 8784-8789

14. Kraeft, S., Traincard, F., Bourdais, J., Mesnildrey, S., Veron, M., and Chen, L. B. (1996) *Exp. Cell Res.* **227**, 63-69

15. Pinon, V. P. B., Millot, G., Munier, A., Vassy, J., Linares-Cruz, G., Capeau, J., Calvo, F., and Lacombe, M. L. (1999) *Exp. Cell Res.* **246(2)**, 355-367

16. Lambeth, D. O., Mehus, J. G., Ivey, M. A., and Milavetz, B. I. (1997) *J. Biol. Chem.* **272**, 24604-24611

17. De La Rosa, A., Williams, R. L., and Steeg, P. S. (1995) *BioEssays* **17**, 53-62

18. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) *Science* **270**, 484-487

19. Zhang, L., Zhou, W., Velculescu, V., Kern, S., Hruban, R., Hamilton, S., Vogelstein, B., and Kinzler, K. (1997) *Science* **276**, 1268-1272

20. Xu, J., Liu, L. Z., Deng, X. F., Timmons, L., Hersperger, E., Steeg, P., Veron, M., and Shearn, A. (1996) *Dev. Biol.* **177**, 544-557

21. Sastre-Gareau, X., Lacombe, M.-L., Jouve, M., Veron, M., and Magdelenat, H. (1992) *Int. J. Cancer* **50**, 533-538

22. Postel, E. H., Berberich, S. J., Flint, S. J., and Ferrone, C. A. (1993) *Science* **261**, 478-480

23. Postel, E. H., and Ferrone, C. A. (1994) *J. Biol. Chem.* **269**, 8627-8630

24. Berberich, S. J., and Postel, E. H. (1995) *Oncogene* **10**, 2343-2347

25. Ji, L., Arcinas, M., and Boxer, L. M. (1995) *J. Biol. Chem.* **270**, 13392-13398

26. Michelotti, E. F., Sanford, S., Freije, J. M. P., MacDonald, N. J., Steeg, P. S., and Levens, D. (1997) *J. Biol. Chem.* **272**, 22526-22530

<footnote n="3">³ F. Agou, unpublished results.</footnote>
27. Wells, R. D. (1988) *J. Biol. Chem.* **263**, 1095–1098

28. Mirkin, S. M., Lyamichev, V. I., Drushlyak, K. N., Dobrynin, V. N., Filippov, S. A., and Frank-Kamenetskii, M. D. (1987) *Nature* **330**, 495–497

29. Simonsson, T., Pecinka, P., and Kubista, M. (1998) *Nucleic Acids Res.* **26**, 1167–1172

30. Albert, T., Mautner, J., Funk, J. O., Hortnagel, K., Pullner, A., and Eick, D. (1997) *Mol. Cell. Biol.* **17**, 4363–4371

31. Michelotti, E. F., Michelotti, G. A., Aronsohn, A. I., and Levens, D. (1996) *Mol. Cell. Biol.* **16**, 2656–2659

32. Hildebrandt, M., Lacombe, M.-L., Mesnildrey, S., and Veron, M. (1995) *Nucleic Acids Res.* **23**, 3858–3864

33. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) *Molecular Cloning: A Laboratory Manual* 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

34. Puglisi, J. D., and Tinoco, I., Jr. (1989) *Methods Enzymol.* **180**, 304–325

35. Karlsson, A., Mesnildrey, S., Xu, Y., Morera, S., Janin, J., and Veron, M. (1996) *J. Biol. Chem.* **271**, 19928–19934

36. Record, M. T., Jr., Lohman, M. L., and De Haseth, P. (1976) *J. Mol. Biol.* **107**, 145–158

37. Lohman, T. M., deHaseth, P. L., and Record, M. T., Jr. (1980) *Biochemistry* **19**, 3522–3530

38. Laue, T. M., Bhaivari, D. S., Ridgeway, T. M., and Pelletier, S. L. (1992) in *Analytical Ultracentrifugation in Biochemistry and Polymer Science* (Harding, S., Rowe, A. J., and Horton, J. C., eds), pp. 90–125, Royal Society of Chemistry Press, Cambridge, UK

39. Schaertl, S. (1996) *FEBS Lett.* **394**, 316–320

40. Webb, P. A., Perisic, O., Mendola, C. E., Backer, J. M., and Williams, R. L. (1995) *J. Mol. Biol.* **251**, 574–587

41. Mesnildrey, S., Agou, F., and Veron, M. (1997) *FEBS Lett.* **418**, 53–57

42. Flinta, C., Persson, B., Jornvall, H., and von Heijne, G. (1986) *Eur. J. Biochem.* **154**, 193–196

43. Postel, E. H., Mango, S. E., and Flint, S. J. (1989) *Mol. Cell. Biol.* **9**, 5123–5133

44. Ha, J. H., Spolar, R. S., and Record, M. T., Jr. (1989) *J. Mol. Biol.* **209**, 801–816

45. Schaertl, S., Konrad, M., and Geeves, M. A. (1998) *J. Biol. Chem.* **273**, 5662–5669

46. Schneider, B., Xu, Y., Sellam, O., Sarfati, R., Janin, J., Veron, M., and Deville-Bonne, D. (1998) *J. Biol. Chem.* **273**, 11491–11497

47. Woodbury, C. P., Jr., and von Hippel, P. H. (1983) *Biochemistry* **22**, 4730–4737

48. Postel, E. H., Weiss, V. H., Beneken, J., and Kirtane, A. (1996) *Proc. Natl. Acad. Sci. U. S. A.* **93**, 6892–6897

49. Battey, J., Moulding, C., Taub, R., Murphy, W., Stewart, T., Potter, H., Lenoir, G., and Leder, P. (1983) *Cell* **34**, 779–787

50. Levens, D., Duncan, R. C., Tomonaga, T., Michelotti, G. A., Collins, I., Davis-Smyth, T., Zheng, T., and Michelotti, E. F. (1997) *Curr. Top. Microbiol. Immunol.* **224**, 33–46

51. Tomonaga, T., and Levens, D. (1996) *Proc. Natl. Acad. Sci. U. S. A.* **93**, 5830–5835

52. Tomonaga, T., Michelotti, G. A., Libutti, D., Uy, A., Sauer, B., and Levens, D. (1998) *Mol. Cell* **1**, 759–764