Excision repair (dimer excision, strand breakage and repair replication) in primary cultures of eukaryotic (bovine) cells

Abstract

Excision repair of ultraviolet radiation damage to DNA in primary cultures of eukaryotic (bovine) cells can be demonstrated by the formation of DNA strand breaks after irradiation, excision of cyclobutyl pyrimidine dimers, and repair replication. Rapidly after irradiation (within 5 min) a steady state is established in which the number of strand breaks (8 000/cell at 100 ergs/ mm 2) and the rate at which bases are inserted by repair replication reach a constant level which is then maintained for several hours. Thereafter the number of strand breaks and the rate of repair replication diminish and 75 to 80% of the dimers are excised in 24 h. On the basis of the close correlation of the kinetics of the various aspects of excision repair, we have suggested that repair is effected by complexes of repair enzymes, each of which performs all steps of excision repair. Such complexes would insert between 70 and 140 bromouracil or thymine bases for each excised photoproduct and spend about 20 min for location of a photoproduct and completion of a repair region. Hydroxyurea did not interfere with rejoining of the strand breaks. Chloroquin, in contrast to reports in E. coli, had no specific effect on excision repair. Immediately after irradiation, cells exposed to high pH and a chelating agent (EDTA) lyse to release DNA more rapidly than control cells. This accelerated release, which may be the consequence of DNA strand breaks or UV damage to membranes and proteins, is lost by about 3 h after irradiation, suggestive of a recovery process distinct from excision repair.