DEVELOPMENT OF THE ARK ASSAY FOR QUANTITATING DNA-PROTEIN CROSSLINK ACCUMULATION AND FANCONI ANEMIA PATHWAY INVOLVEMENT IN THE REPAIR PROCESS
Abstract
DNA-protein crosslinks (DPCs) are a common DNA lesion naturally arising in cells, wherein protein becomes covalently and irreversibly bound to the DNA. Given their excessive size, these adducts present a significant challenge to replication and transcription, thus requiring timely and efficient repair. However, the precise mechanisms involved with processing DPC removal remain unclear. Moreover, current methodologies to quantitate DPC accumulation and removal are restrained by a range of limitations. Here, we describe and discuss a new DPC detection assay – the ARK assay – capable of overcoming the limitations incurred by prior assays. The design, which uses dual chaotropic lysis and anionic denaturation to remove excessive background signal, is premised upon isolating and measuring DPC-associated DNA and free, soluble DNA fragments. We show that the ARK assay effectively detects DPCs induced by a range of agents, and that DPC-defective models exhibit increased DPC accumulation. Functionally, we observe that Fanconi anemia pathway-inactivated cells incur increased DPC accumulation and delayed repair, suggesting a role for the Fanconi anemia pathway in the processing of these deleterious lesions.