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Molecular and Cellular Biology, December 2000, p. 9076-9083, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 and rad53 Lethality

Xiaolan Zhao,1 Bilyana Georgieva,1 Andrei Chabes,2 Vladimir Domkin,2 Johannes H. Ippel,3 Jürgen Schleucher,3 Sybren Wijmenga,3 Lars Thelander,2 and Rodney Rothstein1,*

Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, New York, New York 10032,1 and Department of Medical Biosciences2 and Department of Medical Biochemistry and Medical Biophysics,3 Umeå University, Umeå, SE-90187, Sweden

Received 21 July 2000/Returned for modification 30 August 2000/Accepted 15 September 2000

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.

* Corresponding author. Mailing address: Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, 701 West 168th St., New York, NY 10032. Phone: (212) 305-1733. Fax: (212) 923-2090. E-mail: rothstein{at}cuccfa.ccc.columbia.edu.

Molecular and Cellular Biology, December 2000, p. 9076-9083, Vol. 20, No. 23
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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