High-mobility-group 1 protein mediates DNA bending as determined by ring closures.

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.

Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin.

The mechanism of action of the anticancer compound cis-diamminedichloroplatinum(II) (cisplatin) involves covalent binding to DNA. In an effort to understand the tumor-specific cytotoxicity of such DNA damage, the interactions of these lesions with cellular proteins have been studied. One such protein has been identified as the high-mobility group protein HMG1. Recombinant rat HMG1 binds specifically (dissociation constant 3.7 +/- 2.0 x 10(-7) molar) to DNA containing cisplatin d(GpG) or d(ApG) intrastrand cross-links, which unwind and bend DNA in a specific manner, but not to DNA modified by therapeutically inactive platinum analogs. These results suggest how HMG1 might bind to altered DNA structures and may be helpful in designing new antitumor drugs.

Isolation and characterization of human cDNA clones encoding a high mobility group box protein that recognizes structural distortions to DNA caused by binding of the anticancer agent cisplatin.

Human cDNA clones encoding a structure-specific recognition protein, SSRP1, that binds specifically to DNA modified with cisplatin have been isolated and characterized. The SSRP1 gene maps to human chromosome 11q12. The cDNA clones, obtained by using partial-length cDNAs described previously, predict an 81-kDa protein containing several highly charged domains and a stretch of 75 amino acids 47% identical to a portion of the high mobility group (HMG) protein HMG1. This HMG box most likely constitutes the structure recognition element for cisplatin-modified DNA, with the probable recognition motif being the local duplex unwinding and bending toward the major groove that occurs upon formation of intrastrand cis-[Pt(NH3)2]2+ d(GpG) and d(ApG) cross-links. Although the DNA recognition properties of members of the HMG-box family of proteins have been characterized with respect to their sequence specificity, the present work demonstrates that proteins with this domain can recognize particular DNA structures as well. The Pt-DNA SSRP described here is the human homolog of a recently identified mouse protein that binds to recombination signal sequences [Shirakata, M., Hüppi, K., Usuda, S., Okazaki, K., Yoshida, K. & Sakano, H. (1991) Mol. Cell. Biol. 11, 4528-4536]. These sequences have been postulated to form stem-loop structures, further implicating local bends and unwinding in DNA as a recognition target for HMG-box proteins. Expression analysis in a variety of tissues and cisplatin-resistant cell lines and the inability of cisplatin to induce the message in HeLa cells argue against a direct link between SSRP1 mRNA levels and the response of cells to the drug.