Identification of mammalian DNA repair factors using a reconstituted subcellular system : Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12000–15000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase β-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.