PROJECT SUMMARY Proper maintenance of deoxyribonucleotide triphosphate (dNTP) pools is necessary for high-fidelity DNA replication and repair. Even small changes in the dNTP pools can lead to high rates of mutagenesis, which is commonly seen in human cancers. A key regulator of dNTP pools is ribonucleotide reductase (RNR), the sole enzyme capable of de novo generation of deoxyribonucleotides via radical chemistry. RNRs are conserved across most forms of life, and are split up into three classes based on the cofactor that generates the radical necessary for catalysis. Most of our mechanistic understanding of RNRs comes from class Ia RNRs, which is the class found in humans. The activity of human RNR (HsRNR) is allosterically regulated by the binding of ATP or dATP to the catalytic subunit (α), where the binding of these effectors acts as an on or off switch, respectively. The binding of these effectors also induces the formation of two morphologically identical α6 rings, α6-ATP and α6-dATP. The two hexamers vary in their stability: where only α6-ATP can be disassembled by the radical- generating subunit (β) to form the holoenzyme, whereas α6-dATP is undisturbed by addition of the β subunit. The chemotherapeutic agent clofarabine triphosphate is a dATP-mimic that is hypothesized to allosterically inhibit HsRNR, inducing the formation of α6-dATP-like “persistent hexamers.” These results suggest that targeting allosteric activity sites of HsRNR is a promising approach for development of new anticancer drugs, but the molecular mechanisms underpinning activity regulation have not been fully established. Protein regulators of HsRNR have also been identified, but there is no structural data on the mode of binding of any protein regulator and limited characterization of the molecular mechanism of protein-based regulation of HsRNR. Therefore, we propose studies that aim to answer questions about the molecular mechanisms of activity regulation of HsRNR, using biochemical and biophysical techniques to probe both HsRNR activity regulation via ATP/dATP and also HsRNR activity regulation via protein regulators. The results of this work will provide key details into the activity regulation of HsRNR, along with the first structure of RNR in complex with a protein regulator. This work will be carried out in the lab of Prof. Catherine L. Drennan at the MIT Department of Biology and using the services provided by Dr. Daniel Derege and Dr. Patrick Wintrode of the Mass Spectrometry facility at the University of Maryland: Baltimore’s School of Pharmacy and in collaboration with the laboratory of Dr. Mary Dasso at the National Institutes of Child Health and Human Development.