Ribonucleotide Reductases: Structure and Function

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms providing the monomeric precursors required for DNA replication and repair. The RNRs serve as a paradigm for understanding how Nature has harnessed the enhanced reactivity of protein and nucleotide free radicals with exquisite control to produce deoxynucleotides. The central role of RNRs in nucleic acid metabolism has made them the successful target of two drugs used clinically: 2', 2"-difluoro-2'-deoxycytidine (gemzar) and hydroxyurea. Both compounds interfere with the radical chemistry of the RNRs, the details of which are being investigated in the present proposal. The class I RNRs, require a diferric-tyrosyl radical (Y() cofactor for catalysis. Its function is to initiate the unprecedented long range proton coupled electron transfer (PCET), the radical propagation step, over a 35 A distance. This process occurs between the subunits of the RNR: R1 and R2. Methods to measure this distance with site-specifically attached probes and PELDOR and DQC methods are presented. Methods to study PCET using unnatural amino acids (FnYs with x = 1-4) placed into each subunit by intein mediated protein ligations or orthologous tRNA/tRNA synthetase pairs in conjunction with the translational machinery in vivo are described. Methods to trigger the PCET with light are presented in an effort to detect transient amino acid radical intermediates. Studies of the normal reduction process, mechanism based inhibitors, radical propagation and regulation of the specificity and rate of nucleotide reduction, all require an understanding of the quaternary structure of R1 and its interaction with R2. Biophysical methods (ultracentrifugation, dynamic light scattering, stopped flow fluorescence) using site- specifically placed fluorescent and cross-linking agents within R1 and R2 are presented to address this issue. These studies are essential for achieving our long range goals: to understand how cells biosynthesize and maintain the diferric-Y( cofactor essential for RNR activity and to understand quantitatively the complex layers of regulation that control dNTP pools in vivo. The E. coli RNR and the S. cerevisiae and human RNRs are the focus of our efforts.