IMMUNOLOGICAL PROBING OF INDUCTION AND REPAIR OF 8-METHOXYPSORALEN PHOTOADDUCTS IN DNA FROM FANCONI ANEMIA AND NORMAL HUMAN FIBROBLASTS - QUANTITATIVE-ANALYSIS BY ELECTRON-MICROSCOPY

A direct method of immuno-electron microscopy has been employed to sim ultaneously determine 8-methoxypsoralen (8-MOP) photoinduced monoadduc ts (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 grou ps A and D), and of normal human fibroblasts, following treatment by 8 -MOP and 365 nm radiation. The immuno-reaction with monoclonal antibod y 8G1 was performed on DNA extracted from the cells just after photore action, or after a 24 h repair period, and then denatured. Furan-side MA and also a significant proportion of pyrone-side MA were very effic iently 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 th at FA group A cells are not only impaired in the incision of CL, but a lso of MA. The response of FA group D cells is intermediate between no rmal and FA group A cells.