Nuclear RNR-alpha antagonizes cell proliferation by directly inhibiting ZRANB3

Since the origins of DNA-based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate-limiting role in de novo deoxynucleoside-triphosphate (dNTP) biosynthesis. Paradoxically, the large subunit, RNR-alpha, of this obligatory two-component complex in mammals plays a context-specific antiproliferative role. There is little explanation for this dichotomy. Here, we show that RNR-alpha has a previously unrecognized DNA-replication inhibition function, leading to growth retardation. This underappreciated biological activity functions in the nucleus, where RNR-alpha interacts with ZRANB3. This process suppresses ZRANB3's function in unstressed cells, which we show to promote DNA synthesis. This nonreductase function of RNR-alpha is promoted by RNR-alpha hexamerization-induced by a natural and synthetic nucleotide of dA/ClF/CLA/FLU-which elicits rapid RNR-alpha nuclear import. The newly discovered nuclear signaling axis is a primary defense against elevated or imbalanced dNTP pools that can exert mutagenic effects irrespective of the cell cycle

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition

The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α₂β₂ heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation