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

A DNA repair synthesis assay monitoring nucleotide excision repair (NER) wa s established in cell-free extracts of unicellular alga Chlorella pyrenoido sa using cisplatin- or mitomycin C-damaged plasmid DNA as the repair substr ate. The algal extracts promoted a damage-dependent increase in P-32-dATP i ncorporation after normalization against an internal control. To identify t he proteins responsible for NER, a biotin-labeled duplex 27 mer (2 mu g) ir radiated with or without UV (27 kJ m(-2)) was immobilized on streptavidin-c onjugated agarose beads and incubated with C. pyrenoidosa extracts (50 mu g ) to pull down repair proteins. The extracts post incubation with beads car rying unirradiated 27 mer promoted an eightfold increase in repair synthesi s in plasmid DNA (1 mu g) damaged by 16.8 pmol of cisplatin. The extracts o btained after affinity adsorption with UV-damaged DNA ligand, however, fail ed to repair plasmid DNA treated with cisplatin, reflecting that some prote ins crucial to NER had been sequestered by the damaged 27 mer. A polypeptid e similar to 70-72 kDa in molecular mass was found to bind much more strong ly 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 r ights reserved.