Enhanced binding of the TATA-binding protein to TATA boxes containing flanking cisplatin 1,2-cross-links.
The TATA-binding protein (TBP) is essential for transcription initiation in eukaryotes. TBP recognizes and binds to the minor groove of a consensus sequence, TATAAA, known as the TATA box or TATA element. DNA binding is affected largely by hydrophobic contacts and through the intercalation of two sets of adjacent phenylalanine residues. The resultant duplex is sharply kinked, bending toward the major groove. Inspired by prior structural information showing intercalation of a phenylalanine side chain of a high mobility group (HMG) domain into the site of a cisplatin 1, 2-intrastrand d(GpG) cross-link, a series of DNA probes was prepared with one or two such adducts flanking the TATA box positions at or near the sites of TBP intercalation. The platinum adducts bend the DNA toward the major groove and result in as much as a 175-fold increase in binding affinity of the TBP over the unmodified target sequence. Kinetic studies indicate that the enhanced binding to the modified TATA box is predominantly a consequence of a >30-fold slower dissociation rate of the protein-platinated DNA complex. This work demonstrates that it is feasible to design rationally and to synthesize an enhanced affinity-binding site for a sequence-specific DNA-binding protein by appropriate chemical modification of flanking sequences. It also has implications for the mechanism of action of cisplatin.
Cohen
S M
SM
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Jamieson
E R
ER
Lippard
S J
SJ
D001667:Binding, Competitive / Q000187:drug effects; D002945:Cisplatin / Q000378:metabolism* / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D015195:Drug Design; D007700:Kinetics; D009154:Mutation; D015345:Oligonucleotide Probes; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein; D020079:Terminal Repeat Sequences / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D015345:Oligonucleotide Probes; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C094059:cisplatin-deoxy(guanosine monophosphate guanosine) adduct; D004247:DNA; D002945:Cisplatin