title: 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'-PO(4) 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.
authors.affiliation: AstraZeneca R&D Boston, Waltham, Massachusetts 02451, USA. adam.shapiro@astrazeneca.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Ann E
authors.initials: AE
authors.lastname: Eakin
authors.forename: Grant K
authors.initials: GK
authors.lastname: Walkup
authors.forename: Olga
authors.initials: O
authors.lastname: Rivin
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006193:Haemophilus influenzae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D057166:High-Throughput Screening Assays; D007700:Kinetics; D009243:NAD / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D009243:NAD; D004247:DNA; D011088:DNA Ligases