A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis.
The covalent crosslinking of protein to DNA is a form of DNA damage induced by a number of commonly encountered agents, including metals, aldehydes, and radiation as well as chemotherapeutic drugs. DNA-protein crosslinks (DPCs) are potentially bulky and helix distorting and have the potential to block the progression of translocating protein complexes. To fully understand the induction and repair of these lesions, it will be important to identify the crosslinked proteins involved. To take advantage of dramatic improvements in instrument sensitivity that have facilitated the identification of proteins by proteomic approaches, improved methods are required for isolation of DPCs. This article describes a novel method for the isolation of DPCs from mammalian cells that uses chaotropic agents to isolate genomic DNA and stringently remove noncrosslinked proteins followed by DNase I digestion to release covalently crosslinked proteins. This method generates high-quality protein samples in sufficient quantities for analysis by mass spectrometry. In addition, the article presents a modified form of this method that also makes use of chaotropic agents for promoting the adsorption of DNA (with crosslinked proteins) to silica fines, markedly reducing the DPC isolation time and cost. These approaches were applied to radiation- and camptothecin-induced DPCs.
Barker
Sharon
S
Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2.
Murray
David
D
Zheng
Jing
J
Li
Liang
L
Weinfeld
Michael
M
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents / Q000737:chemistry; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*
D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D004247:DNA