The anticancer activity of cis-diamminedichloroplatinum(II) (cisplatin) arises from its ability to damage DNA, with the major adducts formed being intrastrand d(GpG) and d(ApG) crosslinks. These crosslinks bend and unwind the duplex, and the altered structure attracts high-mobility-group domain (HMG) and other proteins. This binding of HMG-domain proteins to cisplatin-modified DNA has been postulated to mediate the antitumour properties of the drug. Many HMG-domain proteins recognize altered DNA structures such as four-way junctions and cisplatin-modified DNA, but until now the molecular basis for this recognition was unknown. Here we describe mutagenesis, hydroxyl-radical footprinting and X-ray studies that elucidate the structure of a 1:1 cisplatin-modified DNA/HMG-domain complex. Domain A of the structure-specific HMG-domain protein HMG1 binds to the widened minor groove of a 16-base-pair DNA duplex containing a site-specific cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] adduct. The DNA is strongly kinked at a hydrophobic notch created at the platinum-DNA crosslink and protein binding extends exclusively to the 3' side of the platinated strand. A phenylalanine residue at position 37 intercalates into a hydrophobic notch created at the platinum crosslinked d(GpG) site and binding of the domain is dramatically reduced in a mutant in which alanine is substituted for phenylalanine at this position.

Cisplatin exerts its anticancer activity by forming covalent adducts with DNA. High-mobility group (HMG)-domain proteins recognize the major 1,2-intrastrand cisplatin-DNA cross-links and can mediate cisplatin cytotoxicity. The crystal structure of HMG1 domain A bound to cisplatin-modified DNA, further analyzed here, reveals intercalation of a key Phe37 residue. Other published structures of HMG domains bound to DNA, including NHP6A and HMG-D, similarly indicate amino acid side chains intercalating into linear DNA to form a bend. To delineate the importance of such side chain intercalations and further to explore the binding modes of different HMG domains toward prebent DNA structures, site-directed mutagenesis was used to generate HMG1 domain A and domain B mutants. The affinities of these mutant proteins for cisplatin-modified DNA were determined in gel electrophoresis mobility shift assays. The results indicate that intercalating residues at positions 16 or 37 can both contribute to the binding affinity. The data further reveal that the length of the loop between helices I and II is not critical for binding affinity. Footprinting analyses indicate that the position of the intercalating residue dictates the binding mode of the domain toward platinated DNA. Both congruent and offset positioning of the HMG domain with respect to the locus of the cisplatin-induced bend in the DNA were encountered. Packing interactions in the crystal structure suggest how full-length HMG1 might bind to DNA by contacting more than one duplex simultaneously. Taken together, these results demonstrate that cisplatin modification of DNA provides an energetically favorable, prebent target for HMG domains, which bind to these targets through one or more side chain and favorable hydrophobic surface interactions.

The anticancer drug cisplatin is particularly effective against testicular tumors. Although the clinical consequences of cisplatin chemotherapy are well-known, the precise mechanism of action remains elusive. Specific recognition of cisplatin-damaged DNA by a class of proteins containing the high-mobility group (HMG) domain DNA-binding motif could play a role in mediating the cytotoxicity of the drug. This study presents a quantitative investigation of binding of the murine testis-specific high-mobility group protein tsHMG to DNA modified by cisplatin. The binding affinity and specificity of this protein to a site-specific 1,2-d(GpG) cisplatin-DNA intrastrand cross-link in a 20 bp probe were determined. A value for the apparent dissociation constant, Kd(app), of 24 +/- 5 nM was obtained by gel mobility shift assays. Binding competition assays with the corresponding unmodified 20 bp probe gave a ratio (rho) of nonspecific to specific Kd(app) values of 230. A polypeptide containing tsHMG domain A (residues 1-82) was expressed and purified to homogeneity. This domain alone was sufficient for specific recognition of cisplatin-modified DNA with a Kd(app) of 300 +/- 50 nM and a rho of 20, a comparatively high discrimination factor. DNase I interference analysis of the adduct-containing strand revealed that tsHMG binding extends over 14 nucleotides, centered around the platinated bases. The domain A polypeptide protection pattern covers a slightly smaller area of 13 nucleotides. The binding affinity and specificity of tsHMG for cisplatin-modified DNA are exceptional compared to those of other HMG-domain proteins studied previously. The possible relevance of these findings to the mechanism of action of cisplatin is discussed.

The autoantigen La regulates the maturation of RNA polymerase III transcripts by binding to their poly(U) termination signal. The modular protein harbors a Nterminal RNA recognition motif (RRM), RRM1, and in the C-terminal domain, a second, atypical RRM2, in addition to a phosphorylation site, and a putative nucleotide binding site. This study presents a detailed investigation into the RNA 3-end binding properties of La by using binding titration and competition assays with subsequent gel mobility shift analysis. Two truncation mutants containing one (La-RRM1) or both (La-RRM1-RRM2) RNA-binding domains were constructed, overexpressed, and purified. A K d value of 25 ؎ 10 nM for La binding to a nonameric RNA ligand with the oligouridylate recognition sequence was obtained, discriminating with a specificity ratio of ϳ100 for this probe over a RNA ligand with a 3-poly(A) stretch. The N-terminal La-RRM1 region was identified as the major contributor of these properties to La, manifested in a 5-fold lower K d of 5 ؎ 3 nM and a slightly increased specificity ratio of 120 for the RNA ligand. The atypical RRM2 in the C-terminal domain of La has an unprecedented negative effect on 3-end RNA recognition, as indicated by a higher K d value of 90 ؎ 10 nM for the La-RRM1-RRM2 mutant but comparable specificity. Thus the C-terminal regions beyond RRM2 positively modulate the RNA binding affinity of La. Negative regulation, however, occurs through Ser 366 phosphorylation decreasing the binding affinity by 2-fold. ATP had no influence on RNA complex formation. The functional implications of these findings for the mechanism of action of La are discussed.Human La protein (lupus antigen or La/SS-B) was originally identified as a major target of the autoimmune response in patients suffering from the autoimmune diseases Sjögren's syndrome and systemic lupus erythematosus (1). It is a conserved RNA-binding protein that recognizes specifically 3Ј-oligouridylate stretches (2). Associated with all RNA polymerase III transcripts where the 3Ј-UUU-OH sequence is added as the transcription termination signal, La is thought to play a central role in the metabolism of these RNAs by acting as a molecular chaperone that stabilizes and/or structures them for further processing (3). In HeLa cell extracts, the 5Ј-and 3Ј-end processing of tRNA precursors is influenced by La (4), and in vitro, La activity has been suggested in RNA polymerase termination, recycling, and initiation reactions (5-8). Furthermore, a Walker A nucleotide-binding motif in La was postulated to confer ATP-dependent RNA/DNA and RNA/RNA helicase activity (9, 10), but recently, the binding of La to double-stranded nucleic acids was shown to be independent of this site, and La does not appear to unwind double-stranded RNA (11). The Walker A motif has instead been implicated in binding to the 5Ј-triphosphate ends of nascent tRNAs, with the affinity negatively modulated by phosphorylation of Ser 366 (4, 7). Cytoplasmic functions for La have also been described, including facilitatio...

The human orthologue of the Drosophila prune protein (h-Prune) is an interaction partner and regulator of the metastasis suppressor protein NM23-H1 (non-metastatic protein 23). Studies on a cellular breast-cancer model showed that inhibition of the cAMP-specific PDE (phosphodiesterase) activity of h-Prune lowered the incidence of metastasis formation, suggesting that inhibition of h-Prune could be a therapeutic approach towards metastatic tumours. H-Prune shows no sequence similarity with known mammalian PDEs, but instead appears to belong to the DHH (Asp-His-His) superfamily of phosphoesterases. In order to investigate the structure and molecular function of h-Prune, we expressed recombinant h-Prune in a bacterial system. Through sequence analysis and limited proteolysis, we identified domain boundaries and a potential coiled-coil region in a C-terminal cortexillin homology domain. We found that this C-terminal domain mediated h-Prune homodimerization, as well as its interaction with NM23-H1. The PDE catalytic domain of h-Prune was mapped to the N-terminus and shown to be active, even when present in a monomeric form. Our findings indicate that h-Prune is composed of two independent active sites and two interaction sites for the assembly of oligomeric signalling complexes.