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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
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A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis

Sharon Barker1, David Murray, Jing Zheng

  • 1Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2.

Analytical Biochemistry
|August 11, 2005
PubMed

Insights

Researchers developed new methods to isolate DNA-protein crosslinks (DPCs), a type of DNA damage. These techniques enable better identification of proteins involved in DPC formation and repair, aiding in understanding cellular responses to DNA damage.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Covalent crosslinking of protein to DNA forms DNA-protein crosslinks (DPCs), a significant form of DNA damage.
  • DPCs are induced by various agents like radiation, chemotherapeutics, metals, and aldehydes.
  • Identifying proteins involved in DPCs is crucial for understanding their induction and repair.

Purpose of the Study:

  • To develop improved methods for isolating DNA-protein crosslinks (DPCs) from mammalian cells.
  • To generate high-quality protein samples suitable for proteomic analysis, particularly mass spectrometry.
  • To facilitate the identification of proteins covalently bound to DNA.

Main Methods:

  • A novel method utilizing chaotropic agents to isolate genomic DNA and remove non-crosslinked proteins.
  • DNase I digestion to release covalently crosslinked proteins from isolated DNA.
  • A modified method using chaotropic agents for DNA adsorption to silica fines to reduce isolation time and cost.

Main Results:

  • Successful isolation of high-quality protein samples from DNA-protein crosslinks.
  • Demonstrated applicability of the methods to radiation- and camptothecin-induced DPCs.
  • Achieved significant reduction in DPC isolation time and cost with the modified method.

Conclusions:

  • The developed methods provide efficient means for isolating DNA-protein crosslinks (DPCs) for proteomic analysis.
  • These techniques are valuable for identifying proteins involved in DNA damage and repair pathways.
  • The improved isolation methods advance the study of DNA-protein crosslinking and its biological implications.

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