A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

ABSTRACT: Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit ? is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-? assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger ?-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of ? with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-?, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of ? self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

SUBMITTER: Fu Y 

PROVIDER: S-EPMC4271543 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature