Stopped-Flow Fluorescence Studies of HMG-Domain Protein Binding to Cisplatin-Modified DNA<sup>†</sup>

2000-06-24T00:00:00Z (GMT) by Elizabeth R. Jamieson Stephen J. Lippard
High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2−4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein−DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein−dU probe in studying protein−DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.