Open AccessMutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 andrad53 Lethality
Author(s) -
Xiaolan Zhao,
Bilyana Georgieva,
Andrei Chabes,
V. D. Domkin,
Hans Ippel,
Jürgen Schleucher,
Sybren S. Wijmenga,
Lars Thelander,
Rodney Rothstein
Publication year - 2000
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.20.23.9076-9083.2000
Subject(s) - ribonucleotide reductase , biology , mutation , mutant , protein subunit , synthetic lethality , genetics , saccharomyces cerevisiae , amino acid , microbiology and biotechnology , yeast , gene
In budding yeast,MEC1 andRAD53 are essential for cell growth. Previously we reported thatmec1 orrad53 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 theSML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relievedmec1 andrad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed inmec1 andrad53 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 sevensml1 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.