Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.