Detection of specific DNA lesions by a combination of comet assay and FISHin plants

We have studied comet formation on Vicia faba nuclei embedded in agarose an d treated with the endonucleases DNase I (to produce SSBs or DSBs at random sites), Fokl (to produce DSBs preferentially within Fold repeats), or EcoR I (to produce DSBs at random sires bur not within Fokl elements). DNase I-i nduced SSBs were detected when enzyme treatment was followed by alkaline de naturation. DSBs efficiently mediated comet formation using neutral conditi ons. FISH with DNA probes, detecting specific chromosomal domains such as F okl element-containing heterochromatin, NORs, or telomeres, was done on com ets. The distribution of FISH signals between the head and tail of comets i ndicated to which degree these domains were damaged and reflected the distr ibution of cleavage sites far the applied restriction endonucleases within these domains. The data confirmed the expectation that the observed comet f ormation was based on enzyme-specific DNA breakage. (C) 2000 Wiley-Liss, In c.