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.

A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

“…16 the use of a streptavidincoated plate to capture biotinylated, ligated DNA 8,15 requires expensive assay plates and includes a washing step. the chemiluminescence assay of gul et al 14 requires a heating step, and the light output is so brief that the flash reagent must be added to one well at a time just before it is read in a plate reader equipped with an injector, a slow process not amenable to screening large libraries. 6.…”

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

“…Efforts to increase throughput have adopted homogeneous phase assays that use a secondary reaction to detect the product of the ligase reaction (17), but the use of labels and multiple steps increase the incidence of false positive and negative results.…”

“…The methods now used to assay ligase activity—which rely on gel electrophoresis of labeled oligonucleotides—have a limited throughput and are tedious when applied for profiling the activity of the ligase for a family of substrates ( 16 ). Efforts to increase throughput have adopted homogeneous phase assays that use a secondary reaction to detect the product of the ligase reaction ( 17 ), but the use of labels and multiple steps increase the incidence of false positive and negative results.…”

Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

“…LigA is well conserved among eubacterial species, is architecturally and biochemically distinct from the ATP-dependent DNA ligases of eukaryotic cells, and has been found to be essential for bacterial viability wherever examined (13,14,15,17,31). Moreover, the DNA ligation reaction has been dissected mechanistically, mutationally, and structurally (8,20,25,26,33,34,35), and screening assays have been reported for the complete reaction cycle and for individual component steps (2,11,18).DNA ligation activities are essential for multiple DNA processes in replication and repair, including the joining of Okazaki fragments into a continuous strand during chromosomal DNA replication. Enzymatically, DNA ligation proceeds via three successive adenylyl transfer steps (Fig.…”