The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.T he enzyme ribonucleotide reductase (RNR) is a critical cellular factor for the synthesis and control of the deoxynucleoside-5′-triphosphates (dNTPs), the building blocks for DNA replication and DNA repair (1). Specifically, RNR is responsible for the reduction of the ribonucleotides to the corresponding deoxynucleotides. In many well-studied cell systems, like the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, or mammalian cells, this reduction takes place at the level of the nucleoside diphosphates (NDP → dNDP), and each of four separate NDPs (ADP, GDP, CDP, and UDP) can serve as RNR substrate (1, 2). Following reduction, the dNDPs are converted to the corresponding dNTPs by nucleoside diphosphate kinase (NDK) (3). The DNA precursor dTTP is not generated directly through this pathway; instead, it is produced from dCTP via dUTP (4).RNRs have been subdivided into several classes, depending on the type of radical used in the catalytic reaction (1, 5-7). The class Ia RNR, as present in E. coli, yeast, and mammalian cells, employs a tyrosyl radical. Structurally, these RNRs are tetramers composed of a dimer of a large subunit (R1) and a dimer of a small subunit (R2). The large subunits contain the catalytic site and two allosteric regulatory sites (termed specificity site and activity site), whereas the small subunit contains the essential tyrosyl radical. The activity site is located at the N terminus of the R1 subunit and functions as an "on-off" switch: ATP binding leads to an active enzyme, whereas dATP binding inhibits the enzyme. Hence, by monitoring the ATP/dATP ratio, the enzyme aims to ensure an overall dNTP level that is presumably optimal for DNA replication (1, 8).The R1 specificity site, is a binding site for dATP, ATP,...