Kinetic studies of the TATA-binding protein interaction with cisplatin-modified DNA

The TATA-binding protein (TBP) recognizes the TATA box element of transcrip tional promoters and recruits other initiation factors. This essential prot ein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility sh ift assays were performed to study the kinetics of TBP binding both to the TATA box and to cisplatin-damaged DNA in different sequence contexts. TBP b inds with high affinity (K-d = 0.3 nM) to DNA containing site-specific cisp latin 1,2-intrastrand d(GpG) cross-links. The k(on) and k(off) values for t he formation of these TBP complexes are 1-3 x 10(5) m(-1) s(-1) and similar to1-5 x 10(-4) S-1, respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. In electrophoretic mobility shift assay competition assays, cisplatin-damaged DNA extensively sequesters TBP from its natural binding site, the TATA box. Nine DNA probes were prepared to determine the flanking sequence dependence of TBP binding to cisplatin-m odified DNA. TBP clearly displays sequence context selectivity for platinat ed DNA, very similar to but not as dramatic as that of the high mobility gr oup protein HMGB1. When TBP was added to an in vitro nucleotide excision re pair assay, it specifically shielded cisplatin-modified 1,2(GpG) intrastran d cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin.