Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins

The results presented describe the effects of various spectator ligands, at tached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding pro tein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by (Pt(1R,2R-diaminocyclohexane)}(2+), cis-[Pt(NH3)(cyclohexylamin e)}(2+), [Pt(ethylenediamine)}(2+), cis-[Pt(NH3)(cyclobutylamine)}(2+), and cis[Pt(NH3)(2-picoline)}(2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discrimi nate between different platinum-DNA adducts. HMGB1 domain A is the most sen sitive to the nature of the spectator ligands on platinum. The effect of th e spectator ligands on protein binding also depends highly on the base pair s flanking the platinated d(GpG) site. Double-stranded oligonucleotides con taining the AG*G*C sequence, where the asterisks denote the sites of platin ation, with different spectator ligands are only moderately discriminated b y the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly depe ndent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl-2] and cis-[Pt(NH,)(cyclohexy lamine)Cl-2] were also examined. The results suggest that HMGB1 protein lev els influence the cellular processing of cis-[Pt(NH3)(cyclohexylamine)}(2+) , but not [Pt((1R,2R)-diaminocyclohexane)}(2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G *A probes but not with the binding amity of HMGB1a and HMGB1 to platinum-da maged dSAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular protein s.