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

• Published in: Cell reports (Cambridge) 2020-01, Vol.30 (4), p.1235-1245.e4
• Main Authors: Hu, Qianghua, Klages-Mundt, Naeh, Wang, Rui, Lynn, Erica, Kuma Saha, Liton, Zhang, Huimin, Srivastava, Mrinal, Shen, Xi, Tian, Yanyan, Kim, Hyeung, Ye, Yin, Paull, Tanya, Takeda, Shunichi, Chen, Junjie, Li, Lei
• Format: Article
• Language: English
• Subjects: camptothecin; Camptothecin - pharmacology; Cross-Linking Reagents - pharmacology; DNA - metabolism; DNA damage response; DNA Repair - genetics; DNA-Binding Proteins - metabolism; DNA-protein crosslinks; DPC repair; etoposide; Etoposide - pharmacology; Fanconi Anemia - genetics; Fanconi Anemia - metabolism; formaldehyde; Gene Knockout Techniques; Genetic Techniques; HeLa Cells; Humans; K-SDS; RADAR; Topoisomerase I Inhibitors - pharmacology
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2019.12.067

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. [Display omitted] •A sensitive assay for the global measurement of DNA-protein crosslinks (DPCs)•Stringent DPC purification scheme improves DPC detection and minimizes background signals•Assay is capable of both enzymatic and nonenzymatic DPC detections•DPC repair is impaired in Fanconi anemia pathway-deficient cells 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.