Electro-elution-based purification of covalent DNA-protein cross-links

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples. The assay separates proteins in agarose-embedded cell lysates based on their propensity to move through an electric field. Proteins that are covalently cross-linked to DNA are not eluted from the agarose plugs, which can then be analyzed using mass spectrometry or antibody-based staining techniques.Isolation of cross-linked proteins by electro-elution provides an alternative over purification methods that rely on the coprecipitation of DNA and proteins. An assay based on the electrophoresis of whole-cell lysates embedded in agarose plugs separates soluble from immobilized proteins, enabling the purification and the subsequent identification of DNA-protein cross-links.