REPAIR OF CISPLATIN-DNA ADDUCTS BY THE MAMMALIAN EXCISION NUCLEASE

Nucleotide excision repair is one of the many cellular defense mechani sms against the toxic effects of cisplatin. An in vitro excision repai r assay employing mammalian cell-free extracts was used to determine t hat the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA a dduct, is excised by the excinuclease from a site-specifically modifie d oligonucleotide 156 base pairs in length. Repair of the minor inters trand d(G)/d(G) cross-link was not detected by using this system. Prot eins containing the high mobility group (HMG) domain DNA-binding motif , in particular, rat HMG1 anti a murine testis-specific HMG-domain pro tein, specifically inhibit excision repair of the intrastrand 1,2-d(Gp G) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occur red in a reconstituted system containing highly purified repair factor s. These results indicate that HMG-domain proteins cart block excision repair of tile major cisplatin-DNA adducts and suggest that such an a ctivity could contribute to the unique sensitivity of certain tumors t o the drug. The reconstituted excinuclease was more efficient at excis ing tile 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments us ing whole cell extracts [Huang, J.-C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Pi-oc. Natl. Acad. Sci. U.S.A. 91, 1 0394-10398]. This result suggests that structural differences among th e platinated DNA substrates, and not the presence of unidentified cell ular factors, determine the relative excision repair rates of cisplati n-DNA intrastrand cross-links in the whole cell extracts.