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

Abstract

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD+-dependent DNA ligase from Haemophilus influenzae

“…The fluorescence resonance energy transfer assay used for enzyme kinetic assays was described in Shapiro et al [17] and used the same DNA oligonucleotides. Assays were performed in 96-well, flat-bottom black polystyrene plates (Greiner Bio-One, Monroe, NC).…”

“…The ratio change (ΔRatio) was calculated by subtracting the ratio at 0 μM NAD + for each nDNA concentration. ΔRatio values were converted into fraction of DNA ligated using the method described in Shapiro et al [17]. The values of the constants a and b [17] were 0.345 and 0.706, respectively.…”

“…ΔRatio values were converted into fraction of DNA ligated using the method described in Shapiro et al [17]. The values of the constants a and b [17] were 0.345 and 0.706, respectively. Values of fraction ligated were converted into specific activities based on the nDNA and enzyme concentrations.…”

A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

“…31 The described denaturation step could potentially result in many false positives as compounds would bind nonspecifically to the ssDNA oligonucleotides as the temperature is cooling down to RT at a rate of 2°C per minute for a total of 92 min. Other ligation assays using fluorescently labeled DNA substrates have also been reported for bacterial DNA ligases, 32,33 and have been shown to be amenable to miniaturization and used in screening large chemical libraries; however, they do require a denaturation step using 8M urea to separate the fully ligated DNA strand harboring the fluorophore from the other strand template harboring the quencher.…”

“…However, the recent introduction of the PS imaging beads results in ''redshifted'' assay readouts that appear to be relatively insensitive to colored compounds, especially those absorbing in the yellow, red, and blue ranges of the light spectrum. 35 Other inherent limitations of SPA are as follows: (i) the requirements for specific infrastructure to handle radioactive material usage and disposal, which is typically expensive and (ii) the NPE due to the excitation of the fluorophore in the beads by the nonspecific radiation from the tracer when using higher energy radioisotopes such as [ 32 For this purpose, we have successfully developed a scintillation proximity-based assay to screen for inhibitors of the enzymatic activity of Lig4 where our strategy was to block the deadenylation step of the Lig4-AMP complex and potentially resulting in an extra selectivity for actives in chemical libraries. A chemical screen was completed against a library of 5,280 compounds, containing known bioactives and FDA-approved drugs, resulting in four confirmed hits (rabeprazole, cytochalasin A, U73122, and NSC95397) with IC 50 values ranging from 1 to 30 mM ( Table 2); though not particularly potent, these compounds have proven useful nonetheless in followup mechanistic studies and in their potential use as tool compounds.…”