The most frequent DNA adduct made by the anticancer drug ciplatn, the 1,2-tarand d(GpG) crosslink, as well as the minor 1,3-intrasrand d(GpTpG) adduct, were both repaired by an us vitro human excision repair system.Fragments of 27-29 nt containg the platinum damage were excised. The high mobility group (HMG)-domain proteins HMG1 and human mitochondrial rnscription factor specifIcaly inhibited repair of the 1,2-intrd cross-link by the human excision nuclease. These results suggest that the types and levels of E1MG-domain proteins in a given tumor may nfluence the responsiveness of that cancer to cisplatin chemotherapy and they provide a rational basis for the synthesis of new platinum anticancer drug candidates.Cisplatin [cis-diamminedichloroplatinum(II)] is one of the most commonly used anticancer drugs, being particularly effective against testicular tumors (1). Although cisplatin reacts with many cellular components (2), DNA adducts are thought to be the main cytotoxic lesions, and cells deficient in excision repair are extremely sensitive to the drug (3, 4). Recently, it was discovered that proteins with high mobility group (HMG)-domain motifs (5, 6) bind specifically to the major cisplatin-DNA adducts, 1,2-intrastrand d(GpG) and d(ApG) cross-links, which comprise =90o ofthe lesions, but not to the rare 1,3-intrastrand d(GpNpG) cross-links (7,8). Initially, it was thought that the binding of HMG-domain proteins might aid in damage recognition and thus promote repair. When a yeast HMG-domain protein, Ixrl, was deleted, however, the cells became less sensitive to the drug (9). This result demonstrated that HMG-domain proteins can indeed play a role in mediating the cytotoxicity of cisplatin but suggested that repair was inhibited rather than promoted. To evaluate this possibility further, we used two HMGdomain proteins, HMG1 (10) and the human mitochondrial transcription factor (h-mtTFA) (11-13), and DNA fragments containing site-specific platinum adducts to investigate directly the effects of HMG-domain proteins on the excision repair of cisplatin-damaged DNA.Cisplatin adducts as well as other bulky lesions are removed from DNA by an ATP-dependent multisubunit enzyme system called the excision nuclease (excinuclease) (14,15). In humans the excinuclease removes cyclobutane pyrimidine dimers in 27-to 29-nt oligomers by hydrolyzing the 5th phosphodiester bond 3' and the 22nd-24th phosphodiester bonds 5' to the modified base (16,17 (see refs. 8, 20, and 21). Each oligomer had a single cis-diammineplatinum(II) moiety coordinated to N7 of guanine at the sites designated by an asterisk. The oligomer (0.5 ug) was labeled in 50 j4 with [y-32P]ATP (7000 Ci/mmol; 1 Ci = 37 GBq) and mixed with 1.0 tig each of the other seven oligomers, which were phosphorylated (in 100 t4) with nonradioactive ATP.The mixture was precipitated with ethanol and the oligomers were resuspended in 40 1A of annealing buffer, which contained 20 mM Tris HCl (pH 7.4), 50 mM NaCl, and 2 mM MgCl2. The mixture was heated at 700C for 2 min an...

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.

Nucleotide-excision repair is the repair system for removing bulky lesions from DNA. Humans deficient in this repair pathway suffer from xeroderma pigmentosum (XP), a disease characterized by photodermatoses, including skin cancers. At the cellular level, XP patients fail to remove cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimi done photoproducts induced by UV light, as well as other bulky DNA lesions caused by various genotoxic agents. XP cells are not particularly sensitive to ionizing radiation or to alkylating agents that cause mostly nonbulky DNA lesions. Therefore, it has generally been assumed that the human nucleotide-excision repair enzyme (excinuclease) is specific for bulky adducts. To determine the substrate range of human excinuclease we used the highly sensitive excision assay and tested bulky adducts, synthetic apurinic/apyrimidinic sites, N6-methyladenine, 06-methylguanine, and mismatches as potential substrates. We found that all of these "lesions" were removed by human excinuclease, although with vastly different efficiencies.

Synthetic DNA substrates containing an acetylaminofluorene (AAF) adduct at each of the three guanine in the G1G2CG3CC sequence were constructed and tested as substrates for reconstituted E.coli (A)BC excinuclease and human excinuclease in HeLa cell-free extract (CFE). The (A)BC excinulcease repaired the three substrates with relative efficiencies of G1:G2:G3 of 100:18:66 in agreement with an earlier report [Seeberg, E., and Fuchs, R.P.P. (1990) Proc. Natl Acad. Sci. USA 87, 191-194]. The same lesions were repaired by the human excinuclease with the strikingly different efficiencies of G1:G2:G3 as 38:100:68. These results reveal that the human excinuclease is affected by the sequence context of the lesion in a different manner than its prokaryotic counterpart.

Human cell free extracts are capable of carrying out damage-induced DNA synthesis in response to DNA damage by UV, psoralen, and cisplatin. We show that this damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is 'repair synthesis' and not an aberrant DNA synthesis reaction potentiated by DNA deformed by adducts. By comparing the denaturable fraction of psoralen adducted DNA which becomes labeled in the repair reaction to that of terminally labeled DNA (without repair) we have found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach we have obtained preliminary evidence that this in vitro system is capable of removing psoralen crosslinks as well.