Ribonucleotide reductases (RNRs) are responsible for reducing ribonucleotides (NDPs) to deoxyribonucleotides (dNDPs) and are required in all organisms in order to make the monomeric precursors used in DNA replication and repair. This research proposal outlines the development of class I RNR constructs in which the two subunits or a subunit mimic are covalently attached. Incorporation of azido and alkynyl functionality into to R1 and R2 subunits, respectively, allows for their site-specific attachment via a [3+2] cycloaddition. The covalent attachment of the subunits will reduce the conformational flexibility of the amino acid residues believed to be involved in the PCET pathway. This conformation restriction and systematic mutation of the amino acids along the proposed PCET pathway will facilitate the build up of radical species in the construct thereby allowing the proposed radical intermediates to be observed spectroscopically. Observation of the radical species of each of the residues in the pathway will prove their respective involvement in the mechanism of deoxyribonucleotide reduction and allow for the rates of these PCET events to be determined. Studies of the temperature and isotopic dependence of these rates will determine if these processes are limited by protein conformational changes or the PCET process itself. The successful completion of the research outlined in this proposal will provide a detailed mechanistic understanding of the radical transport mechanism in ribonucleotide reductase.
Comparing the mechanism of ribonucleotide reduction in mammalian cells to that in bacterial and viral cells will provide insight into the development of drugs that can specifically target the radical-based intermediates in the bacterial and viral RNR, thereby inhibiting their replication and repair without disrupting the analogous mammalian processes.
