RODENT UV-SENSITIVE MUTANT-CELL LINES IN COMPLEMENTATION GROUPS 6-10 HAVE NORMAL GENERAL EXCISION-REPAIR ACTIVITY

Mammalian nucleotide excision repair is the primary enzymatic pathway for removing bulky lesions from DNA. The repair reaction involves thre e main;steps: (i) dual incisions on both sides of the lesion; (ii) exc ision of the damaged base in an oligonucleotide 24-31 nt in length; (i ii) filling in of the post-excision gap and ligation. We have develope d assays that probe the I individual steps of the reaction. Using thes e methods (assays for incision, excision and repair patch synthesis), we demonstrate that the mammalian excision nuclease system removes bul ky lesions by incising. mainly at the 22nd-25th phosphodiester bonds 5 ' and the 3rd-5th phosphodiester bonds 3' of the lesion, thus releasin g oligonucleotides primarily 26-29 nt in length. The resulting excisio n gap is filled in by DNA polymerases delta and epsilon as revealed by the 'phosphorol thioate repair patch assay'. When these assays were e mployed with cell-free extracts from the moderately UV-sensitive roden t mutants in complementation groups 6-10, we found that these mutants are essentially normal in all three steps of the repair reaction. This leads us to conclude that these cell lines have normal in vitro repai r activities and that the defects in these mutants are most likely in genes controlling cellular functions not directly involved in general excision repair.