The ARK Assay Is a Sensitive and Versatile Method for the Global Detection of DNA-Protein Crosslinks

Abstract

Summary: DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair. : Hu et al. develop a protocol to analyze DNA-protein crosslinking (DPC) damage. Designated the ARK assay, this method outperforms widely used assays by allowing the detection of global DPCs with improved sensitivity and expanded readout. Defective DPC repair is detected in Fanconi anemia mutant cells by this protocol. Keywords: DNA-protein crosslinks, DNA damage response, DPC repair, formaldehyde, camptothecin, etoposide, K-SDS, RADAR