Assaying Uracil-DNA Glycosylase Using Molecular Beacon

DNA glycosylases are a group of specific enzymes that recognize the specific deoxyuradine lesion in DNA and remove them, subsequently initiating base excision-repair (BER). BER is the major pathway that protects cells from nucleotide base damage, Therefore it is of great interest to develop an assay that is able to rapid and reproducible measure DNA glycosylase activities. Usually, the DNA glycosylase activity were assayed by common methods which generally involved the use of radioactively labelled DNA substrates and required polyacrylamide gel electrophoresis (PAGE) to separate the products for qualitive quantitative analysis. Here, a sensitive fluorescent method for assaying DNA glycosylases activity is developed by using molecular beacon (MB) substrate. The measurement has been adaptted to the assay of uracil DNA glycosylases (UDG) by employing a uracil base modified 33 bp MB as substrate. The secondary structure of MB contains a single-stand loop flanked by a short stem that brings the fluorophore and the quencher close...