A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening.
We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.
Woo
Youn-Hi
YH
414 Wartik Laboratory, Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Rajagopalan
P T Ravi
PT
Benkovic
Stephen J
SJ
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis; D015254:DNA Modification Methylases / Q000032:analysis* / Q000378:metabolism*; D015280:DNA Restriction-Modification Enzymes / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D015203:Reproducibility of Results
D015280:DNA Restriction-Modification Enzymes; D004247:DNA; D015254:DNA Modification Methylases; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases