The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a proteinprotein 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 [Sir1 ]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1 27-149 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 silencerbound ORCs in vivo.Chromatin, the protein-DNA complex that comprises eukaryotic chromosomes, varies substantially with chromosomal position, and this structural heterogeneity is fundamental to genome function. A central question in chromosome biology concerns the mechanisms that establish this structural variation in the genome. Silencing of the cryptic mating-type locus HMRa in budding yeast is a powerful model for examining mechanisms that target and confine the formation of a specialized form of chromatin to specific regions of the genome (20,46). Silencing is caused by the formation of "silent" chromatin that is analogous to metazoan heterochromatin, causing heritable, position-dependent transcriptional repression, delayed replication time, and inaccessibility of the chromosomal DNA to a variety of DNA-modifying enzymes (46). Silent chromatin is targeted to HMRa by protein-DNA and protein-protein interactions that require the origin recognition complex (ORC) (20), the evolutionarily conserved multisubunit protein complex best known for its role in the initiation of eukaryotic DNA replication (2). ORC, along with additional sequence-specific DNA-binding proteins Rap1 and Abf1, binds to a small, ϳ150-bp DNA sequence element called the HMR-E silencer. HMR-E is both necessary and sufficient to nucleate assembly of a silent chromatin domain that ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.