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Updated: Jun 17, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
Measurement of DNA interstrand crosslinking in naked DNA using gel-based methods
Konstantinos Kiakos1, Janet M Hartley, John A Hartley
1Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, Paul O'Gorman Building, London, UK.
Abstract:
Bifunctional DNA damaging agents continue to be the mainstay in various chemotherapeutic regimens used in the clinic. DNA interstrand crosslinks are considered to be the critical cytotoxic lesions for the biological activity of such agents. Gel-based electrophoretic assays can efficiently separate denatured single-stranded DNA from double-stranded, covalently-linked DNA resulting from the presence of an interstrand crosslink. The methods described here offer a simple way for the assessment of crosslinking efficiencies of bifunctional agents in both long fragments of DNA (e.g. 1-5 kb) and short oligonucleotide DNA duplexes. As the repair of interstrand crosslinks is a key determinant of cellular and clinical chemosensitivity, these methods can be useful for the characterization and isolation of site-directed adducted substrates for use in subsequent biochemical analysis of cellular recognition and DNA repair processes.
Insights
This study presents gel-based electrophoretic assays to measure DNA crosslinking efficiency. These methods aid in understanding DNA repair mechanisms and improving chemotherapy drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Bifunctional DNA damaging agents are crucial in chemotherapy.
- DNA interstrand crosslinks (ICLs) are key cytotoxic lesions.
- Understanding ICL formation and repair is vital for chemosensitivity.
Purpose of the Study:
- To describe simple gel-based electrophoretic assays for assessing DNA crosslinking efficiencies.
- To enable the evaluation of crosslinking in both large DNA fragments and short oligonucleotide duplexes.
- To provide tools for isolating adducted DNA substrates for further biochemical analysis.
Main Methods:
- Utilizing gel-based electrophoresis to separate single-stranded DNA from double-stranded, covalently-linked DNA.
- Applying the assay to DNA fragments ranging from 1-5 kb.
- Adapting the method for short oligonucleotide DNA duplexes.
Main Results:
- Demonstrated a simple and efficient method for assessing crosslinking efficiencies of bifunctional agents.
- Showcased the assay's versatility across different DNA lengths.
- Validated the utility of the method for preparing substrates for DNA repair studies.
Conclusions:
- The described gel-based assays provide a straightforward approach to quantify DNA interstrand crosslinking.
- These methods are valuable for characterizing DNA damaging agents and their interactions with DNA.
- The techniques can facilitate research into DNA repair pathways, impacting cancer treatment strategies.
