A Rapid Fluorescent In Vitro Assay Suitable for Studying the Kinetics of O6-Alkylguanine Lesion Progression to DNA Inter-Strand Cross-Links and the Kinetics of the Primary Lesion’s Repair

In the author’s opinion, by far the best assay for MGMT activity in cells is the assay developed by Ishiguro, et al. [1-3]. This assay is based on the transfer of radioactive benzyl residues from [benzene- 3H]O6-benzylguanine to MGMT However, this assay is unsuitable for studying the kinetics of the progression of the primary O6-alkylguanine lesion to DNA-DNA inter-strand cross-links, and the kinetics of the primary lesion’s repair by MGMT, because the rate constants involved in the Ishiguro assay means it takes several hours to determine the MGMT activity, whereas many of the dynamic processes we are interested in occur on the time frames of minutes or even seconds.