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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to
Tingyu Wen1, Shubo Zhao2, Julian Stingele2
1Department of Chemistry, Johns Hopkins University, 3400 N. Charles St., Baltimore, Maryland 21218, United States.
Abstract:
The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.
Insights
DNA-protein cross-links (DPC) formed from N7-methyl-2'-deoxyguanosine (MdG) threaten cell survival. Cells lacking SPRTN repair are hypersensitive to DNA damage, highlighting downstream effects of initial DNA lesions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- N7-methyl-2 -deoxyguanosine (MdG) is a major DNA adduct from methylating agents.
- While MdG is persistent, its direct physiological impact is considered minimal.
- MdG can react with histones to form DNA-protein cross-links (DPCMdG), posing a threat to cell survival.
Purpose of the Study:
- To develop a method for quantifying MdG and DPCMdG in nuclear DNA.
- To investigate the contribution of DPCMdG to cytotoxicity induced by methyl methanesulfonate (MMS).
- To determine the role of SPRTN in repairing DPCMdG and cellular response to MMS.
Main Methods:
- Development of a tandem mass spectrometry method for quantifying MdG and DPCMdG.
- Analysis of DNA-protein cross-links in methyl methanesulfonate-treated HeLa cells.
- Assessment of cellular hypersensitivity to MMS in cells with impaired DPC repair.
Main Results:
- A method was established to quantify MdG and DPCMdG in nuclear DNA.
- DPCMdG is formed in less than 1% yield relative to MdG levels in MMS-treated cells.
- DPCMdG contributes to MMS cytotoxicity, and cells lacking SPRTN are hypersensitive to MMS.
Conclusions:
- The downstream effects of initial DNA damage, like DPCMdG formation, have significant biological consequences.
- SPRTN-mediated repair of DPCMdG is crucial for cellular resistance to DNA methylating agents.
- Initial DNA lesions are only the beginning of complex cellular responses to damage.
