Schematic representation of the constructs used in this study. (A) Telomeric repeats are represented as solid arrowheads; the core X as gray rectangle; ARS1 as dotted rectangle; STAR, URA3, TRP1, and the portion of ADH4 used for targeted integration in the VII-L telomere as open rectangles. Arrows above TRP1 and URA3 indicate the orientation of the gene. ACS (solid diamond), B1 (solid triangle), B2 (inverted open triangle), and Abf1 binding site (open circle) are depicted within ARS1 and core X. The Gal4 binding site in GF100 and URA-UAS_GAL-tel is shown as an open rectangle. (B) The consensus B1/ACS sequence (solid rectangle and solid triangle indicate the positions of B1 and ACS) is shown on top. The HMR-E, core X and ARS1 sequences encompassing B1/ACS are shown in capital letters with the consensus bases shown in bold. The mutated sequences in the ACS⁻ and B1⁻ constructs are shown in small letters and are underlined.

ACS-mediated silencing effects in ARS1 and core X. ARS1-URA-tel, GF6, GF44, ARS1(ACS⁻)-URA3-tel, GF6(ACS⁻), GF44(ACS⁻) (diagrams shown on top) were inserted in the VII-L telomere of wild-type (MCM5, W303) and mutant (mcm5-461, orc2-1, orc5-1, cdc45-1, Δsas2, cdc7-1, cdc6-1, and Δsir2) strains, and %FOA^R was determined for each construct/strain combination. Raw data are shown in Supplemental Table 2. (A) The ratio %FOA^R(ACS⁻)/%FOA^RACS as indicated above each graph represents the effect of destruction of ACS in the construct shown on top in the strains shown on the left. In these exponential graphs, values below 1 depict derepression and values above 1 depict increased repression. (B) The ratio %FOA^RGF6(ACS-ΔSTAR)/%FOA^RGF6(ΔSTAR) shows the effect of destruction of ACS in the absence of STAR element.

B1-mediated silencing effects in ARS1 and core X. ARS1-URA-tel, URA-ARS1-tel, GF6, GF44, ARS1(B1⁻)-URA-tel, URA-ARS1(B1⁻)-tel, GF6(B1⁻), and GF44(B1⁻) were inserted in the VII-L telomere of wild type (MCM5, W303) and mutant (mcm5-461, orc2-1, orc5-1, and cdc45-1) strains and %FOA^R was determined for each construct/strain combination. Raw data are shown in Supplemental Table 3. (A) The ratio %FOA^R(B1⁻)/%FOA^RB1 for ARS1-URA-tel and URA-ARS1-tel is plotted to show the effect of destruction of B1 in the strains shown on the left. (B) The ratio %FOA^R(B1⁻)/%FOA^RB1 for GF44 and GF6 is plotted to show the effect of destruction of B1 in the strains shown on the left. (C) The ratio %FOA^RGF6(B1⁻ΔSTAR)/%FOA^RGF6 shows the effect of destruction of B1 in the absence of STAR element.

Mcm5p has antisilencing activity. (A) Wild-type cells (W303) harboring URA3-UAS_GAL-tel (shown on the top) were transformed with plasmids carrying expression cassettes for the GBD-fusion proteins shown on the left. Percentage of FOA^R for each recombinant protein is plotted in the top graph. Raw data are shown in Supplemental Table 5. The ratio %FOA^RGAL4-fusion/%FOA^RGAL4DBD is shown in the bottom graph and indicates the effect of each recombinant protein. Values below 1 depict antirepression activity, whereas values above 1 depict repression activity. (B) LPY1030 cells harboring URA-UAS_GAL-tel (top) and LPY1029 cells harboring URA3-tel (middle) were transformed with plasmids expressing the proteins shown on the left. Percentage of FOA^R was measured for each recombinant protein in each strain and was plotted. Raw data are shown in Supplemental Table 5. The ratio %FOA^RURA3-UAS-tel/%FOA^RURA3-tel (bottom) shows how the effect of these proteins depends on the UAS_GAL site. (C) Wild-type cells (BY4742) and Δhat1, Δgcn5, Δsas2, and Δsas3 isogenic deletion mutants harboring URA3-UAS_GAL-tel (shown on the top) were transformed with plasmids expressing GBD-Mcm5p or GAL4DBD, respectively. Percentage of FOA^R for each recombinant protein was measured. Raw data are shown in Supplemental Table 5. The ratio %FOA^RGAL4-Mcm5p/%FOA^RGAL4DBD indicates the effect of each gene deletion on the antisilencing activity of Mcm5p.

MCM5 has poor insulating activity. W303 cells (A) and W303Δrif1 (B) cells harboring the GF100 reporter (shown on the top) in the VII-L telomere were transformed with plasmids carrying expression cassettes for the GDB-fusion proteins shown on the left. %FOA^R, %TRP+, and %FOA^R&TRP+ cells for each recombinant protein were measured and plotted in the top, middle, and bottom graphs, respectively. Raw data are shown in Supplemental Table 6.

(A) Mcm5p association with telomeres depends on ACS. ChIP with anti-Mcm5p antibodies (Santa Cruz Biotechnology) was performed in the W303/GF44, W303/GF44(ACS⁻), and mcm5-461 strains. Each experiment was performed with parallel controls where cross-linking or primary antibody incubation was omitted. The abundance of different DNAs (shown on the left) was quantified by PCR after confirming linear PCR range for the input samples. The primers for the core X element in GF44 and GF44(ACS⁻) anneal to the plasmid backbone of the integrating constructs and do not amplify genomic core X. core X signals were also confirmed by slot-blot. The signals in the eluates were subtracted by the signals in the final washes and then divided by the signals in the input fractions to produce “net signals.” Finally, all net signals were normalized so that the corresponding values in GF44 are 100% and then compared with the values in W303/GF44(ACS⁻) and mcm5-461. The figure is representative of two independent ChIP experiments. (B) Wild-type (W303), orc2-1 and orc5-1 cells (shown at the bottom top) were released from mitotic arrest and samples were collected after 10, 35, 60, and 75 min (early G1, late G1/S, S, and G2 phases of the cell cycle, respectively) as shown at the bottom. ChIP with anti-Mcm5p antibodies was conducted as described above. The signals in the eluates were subtracted by the signals in the final washes and then divided by the signals in the input fractions and plotted as % enrichment relative to input. The analyzed genomic loci are indicated in the top left corner of each graph. The figure is representative of two independent ChIP experiments.