ANALYSIS OF INCISION SITES PRODUCED BY HUMAN CELL-EXTRACTS AND PURIFIED PROTEINS DURING NUCLEOTIDE EXCISION-REPAIR OF A 1,3-INTRASTRAND D(GPTPG)-CISPLATIN ADDUCT

Nucleotide excision repair by mammalian enzymes removes DNA damage as part of similar to 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion, We analyzed this dual incision react ion at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a clo sed circular duplex DNA substrate. Incisions were formed in the DNA wi th human cell extracts in which DNA repair synthesis was inhibited. Th e nicks were mapped by restriction fragment end labeling and primer ex tension analysis. Principal sites of cleavage were identified at the 9 th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-m er platinated oligonucleotide by hybridization to a P-32-labeled compl ementary DNA probe. Oligonucleotides were formed at the same rate as t he 3' cleavage, suggesting that both incisions are made in a near-sync hronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstitu ted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, a nd a fraction containing ERCC1-XPF and IF7. All of these components we re required in order to observe any cleavage.