Elsevier

Methods in Enzymology

Volume 234, 1994, Pages 122-131
Methods in Enzymology

[12] Oxidative DNA damage: Endonuclease fingerprinting

https://doi.org/10.1016/0076-6879(94)34083-8Get rights and content

Publisher Summary

This chapter describes methods of obtaining cell-free and cellular DNA damage profiles. Two types of assays are used to quantify strand breaks and the incisions of repair endonucleases—a relaxation assay with supercoiled DNA, which is suitable for cell-free DNA, bacterial plasmid DNA, and mitochondrial DNA (mtDNA), and a modified alkaline elution assay, which allows analysis of damage in chromosomal DNA of cultured mammalian cells. Alkylated DNA bases, such as 7-alkylguanine are converted to AP sites and thus become sensitive to exonuclease III and other repair endonucleases. On the other hand, pyrimidine hydrates, which are sensitive to endonuclease III, may eliminate water and become insensitive. DNA damage profiles of oxidative base modifications are obtained by gas chromatography coupled with mass spectrometry in the selected ion-monitoring mode techniques (GC/MS/SIM). GC/MS/SIM allows differentiation among base modifications that are sensitive to the same repair endonuclease. In a combination of the two methods, DNA base modifications excised by repair endonucleases can be analyzed by GC/MS/SIM.

References (14)

  • M. Salditt et al.

    Virology

    (1972)
  • A.J. Fornace

    Mutat. Res.

    (1982)
  • B. Epe et al.

    Chem.-Biol. Interact.

    (1988)
  • B. Epe et al.

    Mutat. Res.

    (1993)
  • L.F. Povirk et al.

    Mutat. Res.

    (1989)
  • M. Dizdaroglu et al.

    Anal. Biochem.

    (1986)
  • J. Hegler et al.

    Carcinogenesis (London)

    (1993)
There are more references available in the full text version of this article.

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