DNA-SEQUENCE CONTEXT AND PROTEIN-COMPOSITION MODULATE HMG-DOMAIN PROTEIN RECOGNITION OF CIPLATIN-MODIFIED DNA

Proteins containing the high mobility group (HMG) DNA-binding domain f orm specific complexes with cisplatin-modified DNA which shield the ma jor intrastrand d(GpG) and d(ApG) crosslinks from excision repair. The molecular basis for the specificity of binding was investigated for t he two isolated domains of HMG1 with a series of 15-bp oligonucleotide s, d(CCTCTCN(1)GG*N2TCTTC).(GAAGAN(3)CCN(4)GAGAGG), where asterisks d enote N-7-modification of guanosine with cisplatin. Alteration of the nucleotides flanking the platinum lesion modulated HMG1domA recognitio n in this series by over 2 orders of magnitude and revealed an unprece dented preference for N-2 = dA > T > dC. The flanking nucleotide prefe rence for HMG1domB interaction with this oligonucleotide series was le ss pronounced and had only a 20-fold range of binding affinities. For the N-1 = N-2 = dA 15-bp probe, 100-fold stronger binding occurred wit h HMG1domA (K-d = 1.6 +/- 0.2 nM) compared to HMG1domB (K-d = 134 +/- 18 nM). The platinum-dependent recognition of the N-1 = N-2 = dA 15-bp probe saturates at 1 equiv of HMG1domA and is highly specific, as evi denced by the 1000-fold decrease in HMG1domA binding affinity for the corresponding unplatinated oligonucleotide. HMG domains were unable to bind specifically to cisplatin-modified DNA-RNA hybrids, revealing th e need for a deoxyribose sugar backbone for specific complex formation with HMG-domain proteins. Protein-DNA contacts which may account for these observed binding preferences are proposed, and potential implica tions for the biological processing of cisplatin-DNA adducts are discu ssed.