Electron microscopy of DNA excision repair patches produced by human cell extracts

To characterize the process by which the mammalian nucleotide excision repair complex interacts with DNA to recognize and repair lesions, we have investigated the size and distribution of repair patches induced by human cell extracts in ultraviolet light-irradiated plasmid DNA. Repair synthesis was carried out in a buffer substituting biotinylated dUTP for dTTP, to allow repair patches to be detected by electron microscopy after streptavidin/colloidal gold labelling. Individual repair events on circular plasmids that had undergone repair synthesis in cell extracts were scored as gold particles bound specifically to irradiated molecules. Samples of over 2000 irradiated and unirradiated plasmids were counted. Repair synthesis at ultraviolet light photoproducts typically replaced about 30 nucleotides, since 69% of patches contained only one particle of 10 nm gold and 24% of patches contained two gold particles (each covering approx. 29 nucleotides). In addition, the ordering of repair events among damaged plasmids closely fitted a Poisson distribution, indicating that repair of lesions is achieved via a non-processive, random diffusion mechanism. This suggests that the repair complex is not intrinsically processive.