Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA.
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.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
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ER
Jamieson
S J
SJ
Lippard
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics; D006609:High Mobility Group Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D000970:Antineoplastic Agents; D015342:DNA Probes; D006609:High Mobility Group Proteins; D011994:Recombinant Proteins; D004247:DNA; D002945:Cisplatin