DNA-protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA-Pt-protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA-protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug.

• MeSH
• DNA Adducts chemistry   MeSH
• Cisplatin chemistry   toxicity   MeSH
• DNA-Binding Proteins drug effects   MeSH
• DNA biosynthesis   drug effects   MeSH
• DNA Repair MeSH
• Antineoplastic Agents chemistry   toxicity   MeSH
• Cross-Linking Reagents chemistry   toxicity   MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• cisplatin-DNA adduct MeSH Browser
• Cisplatin MeSH
• DNA-Binding Proteins MeSH
• DNA MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH
• transplatin MeSH Browser

Negatively supercoiled, relaxed and linearized forms of pSP73 DNA were modified in cell-free medium by cis-diamminedichloroplatinum(II) (cisplatin). The frequency of interstrand cross-links (ICLs) formed in these DNAs has been determined by: (i) immunochemical analysis; (ii) an assay employing NaCN as a probe of DNA ICLs of cisplatin; (iii) gel electrophoresis under denaturing conditions. At low levels of the modification of DNA (<1 Pt atom fixed per 500 bp) the number of ICLs formed by cisplatin was radically enhanced in supercoiled in comparison with linearized or relaxed DNA. At these low levels of modification, the frequency of ICLs in supercoiled DNA was enhanced with increasing level of negative supercoiling or with decreasing level of modification. In addition, the replication mapping of DNA ICLs of cisplatin was consistent with these lesions being preferentially formed in negatively supercoiled DNA between guanine residues in both the 5'-d(GC)-3' and the 5'-d(CG)-3' sites. Among the DNA adducts of cisplatin the ICL has the markedly greatest capability to unwind the double helix. We suggest that the formation of ICLs of cisplatin is thermodynamically more favored in negatively supercoiled DNA owing mainly to the relaxation of supercoils.

• MeSH
• DNA Adducts metabolism   MeSH
• Cisplatin pharmacology   MeSH
• Nucleic Acid Denaturation MeSH
• Electrophoresis, Agar Gel MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Binding, Competitive MeSH
• Nucleic Acid Conformation MeSH
• DNA, Circular metabolism   MeSH
• Sodium Cyanide pharmacology   MeSH
• Molecular Sequence Data MeSH
• Plasmids drug effects   metabolism   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Cross-Linking Reagents metabolism   MeSH
• Restriction Mapping MeSH
• Base Sequence MeSH
• DNA, Superhelical drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Cisplatin MeSH
• DNA, Circular MeSH
• Sodium Cyanide MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH
• DNA, Superhelical MeSH