Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and are thus responsible for controlling the ratios and amounts of dNTP pools essential for DNA replication and repair and the fidelity of these processes. This proposal focuses on the E. Coli and human class Ia RNRs which require two subunits (? and ) to form distinct quaternary structures ((?2)n(2)m), both active and inactive.
The specific aims of this proposal are focused on the 35 oxidation between the tyrosyl radical (Y? in the subunit) and the active site cysteine to a thiyl radical o the ? subunit and understanding the quaternary structures of these RNRs and their interconversions. Both provide unique targets for therapeutic intervention. SA 1focuses on our efforts to understand the long range proton coupled electron transfer (PCET) oxidation (also called the radical transfer (RT) process) in Ec-RNR using our ability to incorporate unnatural amino acids (UAA) site-specifically into each subunit and to use time resolved biophysical methods (stopped flow (SF)-Vis and -fluorescence (Fl) spectroscopies, the rapid chemical quench (RCQ) method, rapid freeze quench (RFQ) paramagnetic resonance methods (multifrequency electron paramagnetic resonance (EPR at 9, 94, 140 and 263 MHz), electron nuclear double resonance (ENDOR) and pulsed electron/electron double resonance (PELDOR) spectroscopies) to study this reversible oxidation process. SAs 2 and 3 focus on our efforts to understand the quaternary structures of the Ec and h-RNRs, the dynamics of their interconversions and the control of these processes by dNTPs and ATP using biophysical methods (SF-Vis and Fl spectroscopies, size exclusion chromatography (SEC), electron microscopy (EM), X-ray crystallography, small angle x-ray scattering (SAXS), dynamic light scattering (DLS) with a team of collaborators). Our understanding of the quaternary structures is essential to thinking about RT and for our long-range goals of designing new types of inhibitors to interfere with this process, with ?/? and ?/ interactions of the active RNRs, and to stabilize ?/? and ?/ interactions of the inactive RNRs. SA I is a collaborative effort with the Nocera laboratory and SA II and III are collaborative effort primarily with the Drennan laboratory.
Ribonucleotide reductases (RNRs) are the target to three clinically used cancer therapeutics (gemcitabine (F2C), clofarabine (ClF) and hydroxyurea (HU)) with a forth, triapine in ongoing clinical trials. F2C is a mechanism-based RNR inhibitor, ClF alters the quaternary structure of the alpha subunit, and HU destroys the essential tyrosyl radical on the beta subunit. Understanding the long-range oxidation of the class Ia RNRs and their quaternary structures, the specific aims of this proposal, will lead to new mechanisms to target RNR for therapeutic intervention.
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