Epstein-Barr virus episomes as targets for cigarette smoke- and gamma-irradiation-induced DNA damage: studies on the EBNA-1 region by a new gene-specific technique

Following our demonstration of cytochrome P450-independent DNA damage induced by aqueous solutions of cigarette smoke in human mucosal cells in vivo, and in a lymphoblastoid cell line, we have developed a new technique to demonstrate gene-region specific DNA damage, with the EBNA-1 gene present in multiple nuclear matrix-attached episomes in Raji cells serving as an amplified target. DNA was extracted from Raji cells treated by gamma-irradiation or aqueous solutions of cigarette smoke; adducted bases or other damage were removed chemically by depurination/alkali treatment. Single-strand breaks induced directly by cigarette smoke as well as DNA cleaved at the site of former adducts were end-labelled either with alpha-[32P]dCTP or with biotin-16-dUTP. With 32P-labelling, a dose-dependent increase in DNA labelling was seen for different concentrations of cigarette smoke; undiluted smoke produced a similar amount of damage as 22.4 Gy of gamma-irradiation. For isolation of DNA regions that contained biotin label at the sites of former damage, DNA was cut by restriction endonucleases and 3-kb-fragments including the target gene, EBV-EBNA-1, were isolated by agarose-gel electrophoresis. Those containing biotin were selected on streptavidin-coated magnetic beads. PCR amplification of the bound DNA revealed EBNA-1 DNA only when cells were pretreated with either cigarette smoke or gamma-irradiation. The presented method thus provides a new approach for detecting gene-specific damage in a readily accessible target, EBV episomes. The method is also potentially applicable for studying single-copy genes such as p53, the types of adducts involved, and quantitative aspects of DNA damage and its repair.