Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of Mec1 and Rad53 lethality

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 th e SML1 open reading frame. Seven mutations were identified that did not aff ect protein expression levels but relieved mec1 and rad53 inviability. Inte restingly, ail seven mutations abolish the SmI1 interaction with Rnr1, sugg esting that this interaction causes the lethality observed in mec1 and rad5 3 strains. The mutant residues all cluster within the 33 C-terminal amino a cids of the 104-amino-acid-long SmI1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic re sonance studies. Moreover, deletions encompassing the N-terminal half of Sm l1 do not interfere with its RNR inhibitory activity. Finally, the seven sm I1 mutations also disrupt the interaction with past Rnr3 and human R1, sugg esting a conserved binding mechanism between SmI1 and the large subunit of RNR from different species.