6,4,4'-TRIMETHYLANGELICIN PHOTOADDUCT IMMUNODETECTION IN DNA - INDUCTION AND REPAIR IN FANCONIS-ANEMIA AND NORMAL HUMAN FIBROBLASTS

6,4,4'-Trimethylangelicin (TMA)-photoinduced monoadducts (MAs) were de tected and quantified on DNA of normal human and Fanconi's anemia (FA) fibroblasts (complementation groups A and D) by immuno-electron micro scopy. TMA-modified DNA was extracted from the cells just after photor eaction, or after a subsequent 24 h repair period, for analysis of the MA processing capabilities of the different cell lines. Unmodified DN A was extracted from the control cells in parallel. The immunoreaction with antibody 7E3 was performed on single-stranded DNA fragments obta ined by heat-formamide denaturation. On single-stranded DNA fragments scanned in the electron microscope, IgC-labeled MA sites appeared as i solated or clustered IgG molecules, which were not homogeneously distr ibuted. The isolated IgG and the different clusters (doublets, triplet s or near-neighbors (within a distance of 250 nucleotides)) were measu red separately for induction frequency and removal. Few interstrand cr oss-links (CLs) were present on X-shape DNA fragments. At time zero, t he 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-excisio n repair capabilities for both MAs (singlets or clusters) and CLs when compared with normal cells. In each cell line, the relative proportio ns 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.