Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I.
A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.
Sun
Daekyu
D
Institute for Drug Development, Cancer Therapy and Research Center, 14960 Omicron Drive, San Antonio, TX 78245-3217, USA. sun@pharmacy.arizona.edu
Urrabaz
Rheanna
R
D019921:Biotinylation; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007553:Isotope Labeling; D008722:Methods; D009841:Oligonucleotides / Q000302:isolation & purification / Q000378:metabolism; D010761:Phosphorus Radioisotopes
D004791:Enzyme Inhibitors; C000607892:LIG1 protein, human; D009841:Oligonucleotides; D010761:Phosphorus Radioisotopes; D011088:DNA Ligases; D000072481:DNA Ligase ATP