Possible involvement of a 72-kDa polypeptide in nucleotide excision repair of Chlorella pyrenoidosa identified by affinity adsorption and repair synthesis assay

A DNA repair synthesis assay monitoring nucleotide excision repair (NER) was established in cell-free extracts of unicellular alga Chlorella pyrenoidosa using cisplatin- or mitomycin C-damaged plasmid DNA as the repair substrate. The algal extracts promoted a damage-dependent increase in P-32-dATP incorporation after normalization against an internal control. To identify the proteins responsible for NER, a biotin-labeled duplex 27 mer (2 mu g) irradiated with or without UV (27 kJ m(-2)) was immobilized on streptavidin-conjugated agarose beads and incubated with C. pyrenoidosa extracts (50 mu g) to pull down repair proteins. The extracts post incubation with beads carrying unirradiated 27 mer promoted an eightfold increase in repair synthesis in plasmid DNA (1 mu g) damaged by 16.8 pmol of cisplatin. The extracts obtained after affinity adsorption with UV-damaged DNA ligand, however, failed to repair plasmid DNA treated with cisplatin, reflecting that some proteins crucial to NER had been sequestered by the damaged 27 mer. A polypeptide similar to 70-72 kDa in molecular mass was found to bind much more strongly to the damaged DNA than to the control DNA after analyzing the proteins bound to the beads by SDS-PAGE, and this polypeptide is believed to play a role in NER in C. pyrenoidosa. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.