6,4,4'-trimethylangelicin photoadduct immunodetection in DNA: Induction and repair in Fanconi's anemia and normal human fibroblasts

6,4,4'-Trimethylangelicin (TMA)-photoinduced monoadducts (MAs) were detected and quantified on DNA of normal human and Fanconi's anemia (FA) fibroblasts (complementation groups A and D) by immuno-electron microscopy. TMA-modified DNA was extracted from the cells just after photoreaction, or after a subsequent 24 h repair period, for analysis of the MA processing capabilities of the different cell lines. Unmodified DNA was extracted from the control cells in parallel. The immunoreaction with antibody 7E3 was performed on single-stranded DNA fragments obtained by heat-formamide denaturation. On single-stranded DNA fragments scanned in the electron microscope, IgC-labeled MA sites appeared as isolated or clustered IgG molecules, which were not homogeneously distributed. The isolated IgG and the different clusters (doublets, triplets or near-neighbors (within a distance of 250 nucleotides)) were measured separately for induction frequency and removal. Few interstrand cross-links (CLs) were present on X-shape DNA fragments. At time zero, the distribution patterns of TMA-photoinduced IgG-labeled MA sites and CLs, and their amount per 10(6) nucleotides, were similar in the three cell lines. After the 24 h repair period, FA cells from two different genetic complementation groups demonstrated impaired incisian-excision repair capabilities for both MAs (singlets or clusters) and CLs when compared with normal cells. In each cell line, the relative proportions of TMA-induced lesions remaining at time 24 h were similar to those initially induced. This implies analogous processing kinetics towards the TMA-phatoinduced clusters of MAs and CLs in a given cell line. (C) 1997 Elsevier Science S.A.