Ribonucleotide reductases (RNRs) catalyze the rate determining step in DNA biosynthesis conversion of nucleotides to deoxynucleotides. Their central role in nucleic acid metabolism has made them the recent target for design of antitumor and antiviral agents. Their unusual metallo cofactors and their function in initiating the radical dependent nucleotide reduction proces by formation of a thiyl radical has been of interest to inorganic and organic chemists alike. 1. Efforts will focus on understanding the trigger for long range coupled electron/proton transfer, approximately 35 Angstrom, between the two subunits of the class I reductases. Efforts will continue to investigate how the class II RNRs accelerate carbon-cobalt bond homolysis by a factor of 1011. 2. Both class I and II RNRs are inactivated by the clinically active compounds gemcitabine and fluoromethylene cytidine di/triphosphates. Efforts will continue to elucidate the mechanism(s) of inactivation and the structures of the observed nucleotide radical intermediate(s). 3. Studies on the assembly mechanism of the class I RNR's diferric tyrosyl radical cofactor will continue using time resolved physical biochemical methods. Efforts to elucidate the structures of two new intermediates will be undertaken. In addition, further characterization of X, the direct precursor of the active cofactor will be undertaken. 4. Our long range goals are to understand the mechanism of cluster assembly of eucaryotic class I RNRs in vivo as well as in vitro and to understand the role of RNRs in both replication and repair. Toward these ends the yeast R1s (one of which is induced by DNA damage) and R2s will be engineered and expressed, the proteins purified, and antibodies generated. Efforts will be made to determine the location of these proteins within the yeast cell. Ultimately we would like to understand how the allosteric regulation of eucaryotic RNRs inside the cell control the fidelity of DNA replication, and the role of RNRs in check point control of the cell cycle.
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