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.
Jamieson
E R
ER
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Lippard
S J
SJ
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