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Stopped-Flow Fluorescence Studies of HMG-Domain Protein Binding to Cisplatin-Modified DNA†
journal contribution
posted on 2000-06-24, 00:00 authored by Elizabeth R. Jamieson, Stephen J. LippardHigh-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.
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activation parametersHMG 1 domainrate constantsdomain proteins bind71 bp platinated DNA analogueHMG domainsplatinated DNA probesanticancer drug cisplatinHMG 1 domain BDNA adductsbinding propertiesHMG 1 proteinsequence contextbinding affinityassociative rateF 37A domaincisplatin adduct modulatescomplexcisplatin mechanismdissociation constants
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