- DNA-protein crosslinks (DPCs), formed by covalent conjugation of proteins to DNA molecule, are toxic DNA lesions created by exposure to physical or chemical insults or faulty enzymatic reactions. Because of their bulky nature and chemical changes to DNA molecule, they interfere with DNA metabolic processing and transcription. With the recent discovery of proteases and nucleases that participate in the repair of such lesions, the development of an accurate biochemical assay for DPC isolation became a priority for further mechanistic understanding of their repair. Here we propose the STAR assay, a novel sensitive, selective, and versatile method for the direct quantification of DPCs, with high sensitivity to physiologically relevant treatment conditions. The assay is efficient in detecting physically, chemically, or enzymatically created adducts. We demonstrate that nuclear localization is required for crosslinking the proteins to DNA molecule. Combining the STAR assay results with Comet assay and western blot confirmed that the repair of DPCs is a two-step process, starting with the removal of protein part of the lesions followed by the repair of the DNA molecule itself. The STAR assay is applicable for studying formation, repair, and biological significance of DPCs, as well as for chemotherapeutics dose management in personalized medicine.