Kinetic studies of the TATA-binding protein interaction with cisplatin-modified DNA.
The TATA-binding protein (TBP) recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. This essential protein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility shift 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 binds with high affinity (K(d) = 0.3 nm) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The k(on) and k(off) values for the formation of these TBP complexes are 1-3 x 10(5) m(-1) s(-1) and approximately 1-5 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-modified DNA. TBP clearly displays sequence context selectivity for platinated DNA, very similar to but not as dramatic as that of the high mobility group protein HMGB1. When TBP was added to an in vitro nucleotide excision repair assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin.
Jung
Y
Y
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Mikata
Y
Y
Lippard
S J
SJ
D001483:Base Sequence; D002945:Cisplatin / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*
D018736:DNA Adducts; D015342:DNA Probes; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C057270:cisplatin-DNA adduct; D002945:Cisplatin