Photoaffinity Labeling Reveals Nuclear Proteins That Uniquely Recognize Cisplatin−DNA Interstrand Cross-Links

The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process cisplatin−DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP[subscript 6], was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSβ, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSβ binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin-resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions.National Cancer Institute (U.S.) (Grant CA34992

Interaction of Metallothionein-2 with Platinum-Modified 5′-Guanosine Monophosphate and DNA

Human metallothioneins (MTs), a family of cysteine- and metal-rich metalloproteins, play an important role in the acquired resistance to platinum drugs. MTs occur in the cytosol and the nucleus of the cells and sequester platinum drugs through interaction with their zinc–thiolate clusters. Herein, we investigate the ability of human Zn7MT-2 to form DNA–Pt–MT cross-links using the cisplatin- and the transplatinmodified plasmid DNA pSP73. Immunochemical analysis of MT-2 showed that the monofunctional platinum–DNA adducts formed DNA–cis/trans-Pt–MT cross-links and that platinated MT-2 was released from the DNA–trans-Pt–MT cross-links with time. The DNA–cis-/trans-Pt–MT cross-links were also formed in the presence of 2 mM glutathione, a strong S-donor ligand. Independently, we used 5′-guanosine monophosphate (5′-GMP) platinated at N7 position as a model of monofunctional platinum-DNA adducts. Comparison of reaction kinetics revealed that the formation of ternary complexes between Zn7MT-2 and cis-Pt–GMP was faster than that of the trans isomer. The analysis of the reaction products with time showed that while the formation of ternary GMP–trans-Pt–MT complex(es) is accompanied by 5'-GMP release, a stable ternary GMP–cis-Pt–MT complex is formed. In the latter complex, a fast initial formation of two Pt–S bonds was followed by a slow formation of an additional Pt–S bond yielding an unusual Pt(II)S3N coordination with N7-GMP as the only N-donor ligand. The ejection of negligible zinc from the zinc–thiolate clusters implies the initial formation of Zn–(μ-SCys)–Pt bridges involving the terminal thiolate ligands. The biological implications of these studies are discussed