A small region of Sir1p governs a Sir1p-ORC interaction. (A) In a previous study (12), our investigators identified mutant versions of Sir1p incapable of silencing by the natural mechanism but capable of silencing HMR when tethered to the locus by a GBD. (B) Sequence of the OIR. Amino acids causing a SRD phenotype when replaced are shown as black letters on a gray background. Previously identified SRD amino acids are designated by dots (12), and SRD amino acids identified in this study are designated by asterisks. Amino acids important for both Sir1p-ORC and Sir1p-Sir4p interactions are shown as white letters on a black background, and amino acids important for a Sir1p-Sir4p interaction are boxed in gray. The two-hybrid boundaries of the OIR are indicated by brackets, while the protease resistance boundaries are designated by braces.

Minimal region of Sir1p required for a two-hybrid Sir1p-Orc1p interaction. (A) The SRD region fused to the GAD was not able to interact with Orc1p in the two-hybrid assay; however, a larger GAD-Sir1(M473-D68) fusion did interact with Orc1p. Cells were plated in 10-fold serial dilutions, starting with 10⁷ cells/ml, on medium lacking histidine to select for an interaction and on complete medium to determine plating efficiency. The James et al. two-hybrid strain (22) containing a SIR2 deletion (CFY932) was used in all two-hybrid experiments. (B) Two-hybrid analysis of GBD-Sir1p fusion proteins defined the minimal OIR of Sir1p. Amino acid boundaries of Sir1p in each experiment are indicated. (C) The GBD-Sir1p fusion proteins examined in panel B were expressed as determined by protein immunoblotting using an anti-GBD antibody (BAbCO).

Specific amino acid substitutions abolish a two-hybrid interaction between GBD-Sir1p(M473-D678) and GAD-Orc1p(L5-V268). Experiments were performed as described in the legend for Fig. 2.

The OIR of Sir1p bound both Orc1p and ORC with the appropriate specificities in vitro. (A) Wild-type and two mutant versions of GST-OIR were bound to glutathione resin and incubated with extracts from Sf9 cells expressing Orc1p and Orc6p. Starting material (Extract), unbound, 0.5 M NaCl wash, and boiled resin fractions were analyzed by protein immunoblotting with antibodies against Orc1p. (B) Affinity experiments were conducted as described for panel A except that extract from Sf9 cells expressing all six ORC subunits was used. Protein immunoblotting was performed with either antibodies against Orc1p or antibodies against Orc2p, as indicated. (C) Affinity experiments were conducted as described for panel A except that extracts from Sf9 cells expressing only Orc2p and Orc5p were used. (D) Affinity experiments were conducted as described for panel A except that a crude yeast cell extract was used. Extract was produced from either a wild-type strain or a strain expressing an Orc1p lacking its N-terminal 235 amino acids. (E) To ensure that similar amounts of wild-type and mutant versions of GST-OIR were used in affinity experiments, 5 μl of each resin was analyzed by SDS-PAGE. The gel was silver stained to visualize the GST-OIR.

The OIR formed a protease-resistant domain. (A) The amino acid sequence of His6-Sir1p_OIR is shown here with potential trypsin cut sites boxed in gray and potential chymotrypsin cut sites boxed in black. Arrows show sites where the OIR is cleaved by trypsin (normal arrow) and chymotrypsin (barbed arrow). R493G and C595R are marked with an asterisk. (B) Coomassie-stained SDS-PAGE analysis of proteolysis products from limited digestion of wild-type or mutant versions of the OIR with trypsin or chymotrypsin. The full-length OIR is labeled with an A on each gel. Trypsin degradation products are labeled as B and C. Note that C is a doublet produced by cleavage after K481 and K482. Chymotrypsin degradation product is labeled with a B.

Conservation of the Sir1p OIR in other yeast species. (A) Alignments of Sir1p orthologs from S. cerevisiae, S. mikatae, S. bayanus, and S. castellii from amino acids M473 to D611, the same region of Sir1p used in biochemical assays. Identical amino acids are boxed, and the boundaries of the SRD region are indicated by black triangles. The OIR extends from the beginning of the SRD region, Y489 (first black triangle) to D611. (B) Two-hybrid examination of Sir1p orthologs. The regions indicated were cloned and tested for the ability to interact with S. cerevisiae Orc1p as described in the legend for Fig. 2A. In the S. castellii-S. cerevisiae SRD swap, S. castellii amino acids 458 to 468 were replaced with S. cerevisiae amino acids 489 to 499. In the reciprocal swap, S. cerevisiae amino acids 489 to 499 were replaced with S. castellii amino acids 458 to 468.

The OIR includes amino acids necessary for a Sir1p-Sir4p interaction. (A) The Sir1p-Sir4p interaction region was mapped by examining GBD-Sir1p fusions containing different regions of Sir1p. (B) Sir1p mutants were tested for the ability to interact with Orc1p and Sir4p in the two-hybrid assay as described in the legend for Fig. 2A. Mutants were divided into three distinct classes based on their interaction defects. (C) Representative mutants from each class were tested for silencing function at a natural silencer (Natural) and when tethered to silencer via the GBD (Tethered). Silencing was measured as the ability of these MATα strains to mate with a MATa lawn and form viable diploids on selective medium. The “natural” silencer used here was the synthetic silencer (32). The synthetic silencer is more sensitive to Sir1p function (12). The relevant genotype of the strain used for natural silencing was MATα HMR-SSa (CFY762), and the strain used for tethered silencing was MATα HMR-SS(GAL4)a (CFY770). The chromosomal copy of SIR1 has been deleted in both strains.

Sir1p-ORC and Sir1p-Sir4p interactions each contribute to Sir1p's ability to bind HMRa in chromatin. (A) Amino acid substitutions representing each of the three classes of Sir1p mutants (Fig. 7) were engineered at the chromosomal SIR1 locus (26) in a MATα HMR-SSa strain (CFY345). Strains expressing the mutant Sir1ps were assayed for silencing function by examining 10-fold serial dilutions for the ability to form diploids with a MATa lawn on selective medium. An asterisk indicates a strain used for the ChIP assays shown in panel C. (B) Mutant and wild-type Sir1p-3xHA were expressed at similar levels as measured by protein immunoblotting with anti-HA (by IP followed by immunoblotting). (C) Wild-type and mutant Sir1p binding to HMRa was examined using ChIP assays with anti-HA. Two reaction mixtures, using 2 and 4 μl of template, were set up for each immunoprecipitate (IP) and total sample.