Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

Abstract

Repair of DNA damage is a critical survival mechanism that impacts susceptibility to a variety of human diseases and represents a key target for cancer therapy. A major barrier to applying this knowledge in research and clinical translation has been the lack of efficient, quantitative functional assays for measuring DNA repair capacity in living primary cells. To overcome this barrier, we recently developed a technology termed fluorescence multiplex host cell reactivation (FM-HCR). We describe a method for using standard molecular biology techniques to generate large quantities of FM-HCR reporter plasmids containing site-specific DNA lesions and using these reporters to assess DNA repair capacity in at least six major DNA repair pathways in live cells. We improve upon previous methodologies by i) providing a universal workflow for generating reporter plasmids, ii) improving yield and purity to enable large-scale studies that demand milligram quantities, and iii) reducing preparation time more than 10-fold.