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Molecular and Cellular Biology, February 2009, p. 687-702, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.00202-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1{triangledown}

Zhonggang Hou,1 John R. Danzer,1 Liza Mendoza,1 Melissa E. Bose,2 Ulrika Müller,1 Barry Williams,3 and Catherine A. Fox1,2*

Department of Biomolecular Chemistry,1 Laboratory of Genetics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706,2 Departments of Zoology and Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 488243

Received 7 February 2008/ Returned for modification 7 April 2008/ Accepted 10 November 2008

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir127-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir127-149 in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.

* Corresponding author. Mailing address: Department of Biomolecular Chemistry, 587 MSC, 1300 University Ave., University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532. Phone: (608) 262-9370. Fax: (608) 262-5253. E-mail: cfox{at}wisc.edu

{triangledown} Published ahead of print on 24 November 2008.

Molecular and Cellular Biology, February 2009, p. 687-702, Vol. 29, No. 3
0270-7306/09/$08.00+0     doi:10.1128/MCB.00202-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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