DNA Sequence Context and Protein Composition Modulate HMG-Domain Protein Recognition of Cisplatin-Modified DNA

Abstract

Proteins containing the high mobility group (HMG) DNA-binding domain form specific complexes with cisplatin-modified DNA which shield the major intrastrand d(GpG) and d(ApG) cross-links from excision repair. The molecular basis for the specificity of binding was investigated for the two isolated domains of HMG1 with a series of 15-bp oligonucleotides, d(CCTCTCN1G*G*N2TCTTC). (GAAGAN3CCN4GAGAGG), where asterisks denote N7-modification of guanosine with cisplatin. Alteration of the nucleotides flanking the platinum lesion modulated HMG1domA recognition in this series by over 2 orders of magnitude and revealed an unprecedented preference for N2 = dA > T > dC. The flanking nucleotide preference for HMG1domB interaction with this oligonucleotide series was less pronounced and had only a 20-fold range of binding affinities. For the N1 = N2 = dA 15-bp probe, 100-fold stronger binding occurred with HMG1domA (Kd = 1.6 +/- 0.2 nM) compared to HMG1domB (Kd = 134 +/- 18 nM). The platinum-dependent recognition of the N1 = N2 = dA 15-bp probe saturates at 1 equiv of HMG1domA and is highly specific, as evidenced 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 the 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 implications for the biological processing of cisplatin-DNA adducts are discussed.

Interaction of FACT, SSRP1, and the High Mobility Group (HMG) Domain of SSRP1 with DNA Damaged by the Anticancer Drug Cisplatin

“…The HMG domains of HMG1, HMG2, SSRP1, and other HMG-domain proteins that bind cisplatin-damaged DNA are likely to be the key elements in protein recognition of cisplatin-DNA lesions. Evidence supporting this conclusion has been described for both HMG domains of HMG1 (27,40). To determine whether the HMG domain of SSRP1 is sufficient for cisplatin-modified DNA binding, a fragment corresponding to residues 539 -614 was expressed and purified from Escherichia coli.…”

“…Radiolabeled probes were purified with G25 Quick Spin columns (Roche Molecular Biochemicals), ethanolprecipitated, and quantitated by scintillation counting (Beckman LS 6500). The radiolabeled 15-bp oligonucleotide duplexes AG*G*A (d(CCTCTCAG*G*ATCTTC)/d(GAAGATCCTGAGAGG), where asterisks denote the sites of cis-diammineplatinum(II) cross-linking via the N-7 positions of adjacent guanines and AGGA, the unplatinated analog) were prepared according to published procedures (27). Double-stranded 156-bp probes were assembled from six oligonucleotides of lengths 87 nt (fragment 1), 12 nt (fragment 2), 57 nt (fragment 3), 52 nt (fragment 4), 24 nt (fragment 5), and 80 nt (fragment 6) as described previously (28,29).…”

Effects of Spectator Ligands on the Specific Recognition of Intrastrand Platinum-DNA Cross-links by High Mobility Group Box and TATA-binding Proteins

“…Thus, HMGB1b does not discriminate as well between different Pt-DNA lesions when compared with HMGB1a. HMGB1b binds the dsAG*G*C flanking sequence better than dsTG*G*A, on all probes (32). There are fewer differences in binding affinity for dsAG*G*C than dsTG*G*A probes, further demonstrating the influence of the flanking sequence for protein binding.…”

“…Electrophoretic Mobility Shift Assays (EMSAs)-EMSAs for HMGB1a and HMGB1b were performed as described previously (32,33). Protein (30 nM for HMGB1a or 340 nM for HMGB1b) and DNA probe (12-17 nM, 5000 -10000 cpm/l) were incubated on ice for 30 min in a solution containing 10 mM HEPES (pH 7.5), 10 mM MgCl 2 , 50 mM LiCl, 100 mM NaCl, 1 mM spermidine, 0.2 mg/ml bovine serum albumin, and 0.05% Nonidet P-40 (15-l reaction mixtures).…”

“…Previous data revealed that the affinity of HMG box proteins for cisplatin-modified DNA depends on the base pairs immediately flanking the platinum adduct (32,33). Such flanking sequence dependence is especially prominent in the case of HMGB1a, which has a binding affinity that varies by several orders of magnitude for cisplatin-DNA adducts with different flanking sequences.…”