A high-throughput assay for the adenylation reaction of bacterial DNA ligase.
DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.
Miesel
Lynn
L
Schering-Plough Research Institute, Kneilworth, NJ 07033, USA.
Kravec
Cynthia
C
Xin
Alan-Tianpei
AT
McMonagle
Patricia
P
Ma
Sarah
S
Pichardo
John
J
Feld
Boris
B
Barrabee
Ellen
E
Palermo
Robert
R
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D009243:NAD / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting; D019809:Streptavidin
D004791:Enzyme Inhibitors; D008024:Ligands; D009243:NAD; D000249:Adenosine Monophosphate; D001710:Biotin; D000255:Adenosine Triphosphate; D004247:DNA; D019809:Streptavidin; D011088:DNA Ligases; C120640:DNA ligase (NAD); D000241:Adenosine