+, binding; +/, weak binding; -, lack of binding. change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated Dehydrocholic acid in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations. Ribonucleotide reductases (RNRs) provide the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs) for DNA synthesis and repair (37). RNRs catalyze the controlled reduction of all four Dehydrocholic acid ribonucleotides (NTPs) to maintain a balanced pool of dNTPs during the cell cycle and may constitute a rate-limiting step in chromosomal replication initiation (20). In prokaryotes RNR activity is controlled at two main levels. Nucleoside and deoxynucleoside triphosphate effector molecules allosterically regulate enzyme activity and specificity (36), while equally important, though less well understood, is genetic regulation of enzyme activity (42). These processes enable the cell to rapidly adapt to changes in the intracellular replication machinery, to ensure faithful DNA replication and repair, and to respond to changes Dehydrocholic acid brought about by environmental factors, such Dehydrocholic acid as oxygen tension and oxidative stress agents (16,17). Strict control of RNR activity and dNTP pool sizes is important since pool imbalances cause replication anomalies, mutations, and genome instability (10,17,35,41,49). Three major classes of RNRs have been characterized (36). Class I RNRs are oxygen-dependent enzymes that occur in eubacteria, eukaryotes and some viruses, class II RNRs are oxygen-independent enzymes confined to bacteria, archaea, and a few unicellular eukaryotes, and class III RNRs are oxygen-sensitive enzymes present in anaerobes. Despite significant differences in their structures and in cofactor requirements, all three classes of RNRs share similar catalytic mechanisms (13,26,36,37). In prokaryotes class I reductases comprise two main subgroups. Class Ia RNRs are encoded in operons containingnrdAandnrdBgenes that specify the NrdA (R1) subunit, possessing catalytic and allosteric regulatory functions, and the NrdB (R2) subunit, possessing radical-generating activity. Class Ib RNRs are encoded in operons containingnrdEandnrdFgenes that code for the corresponding subunits NrdE and NrdF, respectively. Class Ia and class Ib RNRs share many biochemical features, although their protein subunits have limited sequence identity. Both require oxygen for generation of a tyrosyl radical stabilized by an iron center, which transfers the radical to an active-site cysteine of NrdA or NrdE. They differ in that class Ia RNRs, but not class Ib RNRs, contain in the N-terminal part of NrdA an effector activity site that enables allosteric regulation by ATP/dATP (13,26,36,37). Class II RNRs are encoded by thenrdJgene and use coenzyme B12(adenosylcobalamin) to generate a transient 5-deoxyadenosyl radical. The cofactor fulfills the function of the radical generating subunit in class I enzymes. NrdJ consists of a single polypeptide and is considered to be the simplest of the RNRs. Class III RNRs are encoded bynrdD, which occurs in an operon containingnrdG, coding for a specific activase that usesS-adenosylmethionine to create a stable oxygen-sensitive glycyl radical close to the active site of NrdD. Allosteric regulation of RNRs is mediated by the binding of nucleoside and deoxynucleoside triphosphate effectors to two distinct sites, a specificity site that regulates substrate specificity and an activity site that regulates overall enzyme activity (36). Effectors induce specific Dehydrocholic acid conformational changes in protein structure that modulate enzyme activity, however the molecular mechanisms are not well understood. Bacterial class Ia RNRs (and some class III RNRs) typically contain an effector binding site for regulating overall activity. Binding of ATP to that domain stimulates activity, whereas binding Cd63 of dATP inhibits activity. In contrast, class Ib RNRs (and many class II RNRs) lack the activity site. The crystal structure ofE. coliNrdA complexed with a nonhydrolyzable ATP analogue [AMPPNP adenosine 5-(–imido)-triphosphate] established that the activity site lies in a sequence of approximately 100 amino acids, located at the N-terminal portion of the molecule, which forms a cleft with a four-helix bundle covered by a three-stranded mixed -sheet (14,48). Aravind et al. first coined the term.