Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Abstract

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Scheme 1. Schematic representation of the reaction mechanism of NAD⁺-dependent ligases

(a) Formation of an enzyme–adenylate complex and NMN. (b) Activation of 5′-phosphate at the site of a nick in the DNA substrate. (c) Nick closure in which a covalent bond is formed between an adjacent 3′-hydroxy group and activated 5′-phosphate in duplex DNA structures with the release of AMP.

Scheme 2. Schematic representation of the HPA for S. aureus DNA ligase

After ligation of the AE-labelled DNA substrate, the AE label is rendered less susceptible to alkaline hydrolysis. The label from non-ligated DNA is preferentially hydrolysed by mild alkaline stop/selection reagent [360 mM Na₂B₄O₇ containing 240 mM HCl and 0.07% (v/v) Triton X-102, pH 8] with incubation at 40 °C in a water bath or heating block. Addition of 0.5 M NaOH containing 0.3% (v/v) H₂O₂ results in the decomposition of the AE label from ligated DNA and release of light at 430 nm (chemiluminescence).

Figure 1. Coomassie-Blue-stained SDS/PAGE gel of the final purified S. aureus DNA ligase

Protein was expressed and purified as described in the Materials and methods section. The positions and sizes (in kDa) of the of marker proteins are shown on the left. Lane 1, protein size markers; lane 2, 2 μg of total protein. The band at approx. 70 kDa is that of S. aureus DNA ligase.

Figure 2. (a) Demonstration of the linear dependence of chemiluminescence (RLU) on AE-labelled DNA substrate concentration in the absence of S. aureus DNA ligase (•) and presence of 50 pM S. aureus DNA ligase (^) and (b) kinetics of chemiluminescence (RLU) generation

(a) A linear relationship was shown to exist for the concentration of AE-labelled DNA substrate up to 10 nM and approx. 2×10⁵ RLU in the presence (50 pM) and absence of S. aureus DNA ligase. Reactions were carried out in volumes of 20 μl in Hepes (pH 7.4) assay buffer. Chemiluminescence generated upon addition of 20 μl of a solution of flash reagent was detected over a 2 s period. The calibration curve constructed using the closed circles in (a) was used in converting RLU into AE label concentration and hence that of substrate/product. (b) Reaction of AE-labelled DNA substrate in Hepes (pH 7.4) assay buffer with chemiluminescence-generating flash reagent results in the release of light at 430 nm. The concentration of AE-labelled substrate investigated was in the 0.1–10 nM range. In all cases, the lifetime of the emitted light was under 2 s.

Figure 3. (a) Time-course of the hydrolysis of the AE label on unligated (•) and ligated (▪) DNA substrate and (b) the dependence of signal/background of the chemiluminescent HPA for NAD⁺-dependent S. aureus ligase

(a) Substrate or product (10 nM) in Hepes (pH 7.4) assay buffer were incubated in mild alkaline stop/selection reagent at 40 °C in a water bath or heating block. The AE label content of samples at various times was quantified by measuring the amount of chemiluminescence (RLU) generated upon reaction with chemiluminescence-generating flash reagent. Hydrolysis of the AE label in substrate by the stop/selection reagent is exponential in nature, but is essentially fully protected in product. (b) Values for signal/background ratio at regular intervals were calculated from the chemiluminescence (RLU) of unligated (•) and ligated (▪) AE-labelled DNA substrate from (a). There is an increase in the signal/background ratio over time, and a steady value of 200:1 after 60 min of incubation with stop/selection reagent was achieved.

Figure 4. Time course of S. aureus DNA ligase catalysis of the AE-labelled DNA substrate using the HPA

The assay was carried out using 0.5 nM enzyme and 10 nM AE-labelled DNA substrate in Hepes (pH 7.4) assay buffer. Complete ligation of the AE-labelled substrate is obtained after approx. 60 min of incubation. Upon full ligation of 10 nM AE-labelled substrate, approx. 2×10⁵ RLU were obtained upon analysis, confirming complete ligation.

Figure 5. Demonstration of the adherence to the Michaelis–Menten equation of (a) catalysis of NAD⁺ and (b) ligation of the AE-labelled DNA substrate by S. aureus DNA ligase

(a) Assays were carried out in Hepes (pH 7.4) assay buffer containing 10 nM AE-labelled DNA substrate and 50 pM enzyme, with variation in NAD⁺ concentration between 2 and 45 μM. The points are experimental and the continuous lines correspond to v_i=V_max[S]₀/(K_m+[S]₀) with K_m=2.75±0.1 μM and V_max=(6.20±0.05)×10⁻¹³ M/s. (b) Assays were carried out in Hepes (pH 7.4) assay buffer containing 25 μM NAD⁺, 50 pM enzyme, with variation in the concentration of AE-labelled DNA between 1 and 10 nM. The points are experimental and the continuous lines correspond to v_i=V_max[S]₀/(K_m+[S]₀) with K_m=2.5±0.2 nM and V_max=(6.15±0.05)×10⁻¹³ M/s.

Figure 6. pH-dependence of (a) k_cat/K_m and (b) k_cat for S. aureus DNA-ligase-catalysed ligation of the AE-labelled DNA substrate

These studies were carried out in the following assay buffers at room temperature: sodium acetate (pH 5.2 and 5.6), Hepes (pH 6.0, 6.5, 7.1 and 7.4) and Tris/HCl (pH 8.0). Reactions were initiated by the addition of enzyme (50 pM final enzyme concentration) to solutions of buffer containing substrate (2–10 nM). The points are experimental and the lines are theoretical for the single ionization equation k=k~/(1+[H⁺]/K_a), where k=k_cat or k=k_cat/K_m and k~=k~_cat or k~_cat/K~_m (pH-independent kinetic parameters) (a) pK_a=7.1±0.1, k~_cat/K~_m=(4.1±0.1)× 10⁵ M⁻¹·s⁻¹ and (b) pK_a=6.6±0.1, k~_cat=0.009±0.001 s⁻¹.

Figure 7. Structures of (1) doxorubicin and (2) chloroquine diphosphate, two inhibitors of S. aureus DNA ligase

The IC₅₀ values for inhibition of S. aureus DNA ligase have been determined against these inhibitors using the HPA. For (1), IC₅₀=3.30±0.86 μM; for (2), IC₅₀=1.40±0.07 μM.