Immunological probing of induction and repair of 8-methoxypsoralen photoadducts in DNA from Fanconi anemia and normal human fibroblasts: Quantitative analysis by electron microscopy

Abstract

A direct method of immuno-electron microscopy has been employed to simultaneously determine 8-methoxypsoralen (8-MOP) photoinduced monoadducts (MA) and interstrand cross-links (CL), their relative localization along the DNA molecule, and their removal. It has been applied to DNA from cultures of Fanconi anemia (FA) fibroblasts (complementation groups A and D), and of normal human fibroblasts, following treatment by 8-MOP and 365 nm radiation. The immuno-reaction with monoclonal antibody 8G1 was performed on DNA extracted from the cells just after photoreaction, or after a 24 h repair period, and then denatured. Furan-side MA and also a significant proportion of pyrone-side MA were very efficiently immuno-detected. Only 1–2% CL were IgG-labeled. This is why CL were directly visualized and quantified on denatured DNA from the same cellular samples as used for immuno-detection. Results demonstrate that FA group A cells are not only impaired in the incision of CL, but also of MA. The response of FA group D cells is intermediate between normal and FA group A cells.