In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.Ribonucleotide reductase (RNR) is a highly conserved enzyme that catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs, the rate-limiting step of deoxynucleoside triphosphate (dNTP) formation, and DNA synthesis. Its activity directly affects the balance and the levels of the dNTP pools and subsequently genetic stability (29). Due to its vital importance, RNR is tightly regulated by both cell cycle and environmental cues. At S phase and after DNA damage, RNR activity is up-regulated to provide sufficient and balanced dNTP pools for DNA replication and repair. Mutations interfering with this regulated increase in RNR activity in yeast and humans can lead to growth defects and sensitivity to DNA-damaging agents (12,32). RNR activity is also subjected to negative regulation, which is equally important. This is underscored by the observation that overexpression of a small subunit of yeast or human RNR in yeast cells causes chromosome instability (27). Presumably, the deleterious effects of rampant RNR activity may be due to decreased DNA polymerase fidelity caused by excess dNTP levels and, at the same time, diminished NTP levels, which may interfere with RNA synthesis and numerous ATP/ GTP-dependent cellular processes.Currently, two mechanisms for RNR regulation are known. First, RNR is under allosteric control. In most organisms, the RNR holoenzyme is a tetramer composed of two distinct subunits (␣ 2  2 ), both of which contribute to the enzymatic activity. However, only the large subunit contains two allosteric sites: one regulates the balance among the four dNTP pools, and the other regulates feedback inhibition by monitoring the dATP/ ATP ratio and modulating overall RNR activity accordingly (20). Second, RNR is subjected to transcriptional regulation. In the budding yeast, transcription of th...