Volume 30, Issue 2, Pages 156-166 (April 2008) An ARS Element Inhibits DNA Replication through a SIR2-Dependent Mechanism  Amber Crampton, FuJung Chang, Donald L. Pappas, Ryan L. Frisch, Michael Weinreich  Molecular Cell  Volume 30, Issue 2, Pages 156-166 (April 2008) DOI: 10.1016/j.molcel.2008.02.019 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Chromosome III and VI Origins Inhibited by SIR2 (A) Plasmid loss rates for chromosome III ARS elements in SIR2 and sir2Δ strains (±SEM). (B) Growth of cdc6-4 sir2Δ (M922) with selection for chromosome VI ARS plasmids at 25°C and 37°C. (C) Plasmid loss rates (±SEM) for ARS603 and ARS606 indicates they are inhibited by SIR2. Strains were WT (W303-1A), sir2Δ (M620), cdc6-4 (M386), and cdc6-4 sir2Δ (M922). (D) ARS305 and ARS315 are not inhibited by silent chromatin formation. ARS305, ARS315, and ARS317 plasmid stabilities (±SEM) were determined in WT (W303-1A), sir2Δ (M2126), and sir3Δ (M2154) backgrounds. Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 SIR2 Inhibits Pre-RC Formation for Chromosomal Origins ARS305, ARS603, and ARS606 (A) Primer design. (B) Cells were arrested in G2/M with nocodazole at 25°C, shifted to 37°C, and then released at 37°C into α factor. (C) Mcm2p ChIP at t = 0, 60, and 90 min after release indicates that MCM loading is defective in cdc6-4 cells but is restored at ARS305, ARS603, and ARS606 in the cdc6-4 sir2Δ mutant. WT (W303-1A), cdc6-4 (M386), and cdc6-4 sir2Δ (M940). Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Analysis of ARS305 and ARS315 Origin Structures Reveals an Inhibitory Sequence Element, IS The percentage of cells containing ARS305 (A) or ARS315 (B) linker scan plasmids are shown after growth in nonselective medium for a constant number of generations (±SEM). Two linker scan mutants disrupting the 10/11 (ARS305) or 11/11 (ARS315) match to the ACS were not able to transform W303-1A and define the A element. The linker scan mutations are indicated in Figures S3 and S4, and the corresponding plasmid loss rates per generation are listed for all data points in Table S4. Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 The Inhibitory Element IS Requires SIR2 for Its Activity and Is Contextual (A) ARS305 and ARS315 plasmid loss rates per generation (±SEM) were determined in WT (W303-1A), sir2Δ (M620), cdc6-4 (M386), and cdc6-4 sir2Δ (M922) strains. Loss rates for wild-type ARS305 (pRF4) and ARS315 (pAC25) plasmids are shown as dark blue bars; light blue bars indicate loss rates for the same plasmids with SalI linker mutations in the IS elements (ARS305 +171, Figure S3; ARS315 +187, Figure S4). (B) Chimeric ARS1-ARS305 origin plasmids (diagrammed on the left) were measured for their plasmid loss rates (±SEM) (right). (C) The distance of the IS element with respect to B2 is important. A 44 bp insertion and various deletions were constructed between the B2 and IS elements of ARS305. The effect on plasmid stability (±SEM) was measured with the wild-type IS element (+IS) and a SalI linker scan mutation in IS (−IS). IS mutation is +171, Figure S3. Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Histone H4 K16Q Suppresses cdc6-4 and mcm2-1 Pre-RC Temperature-Sensitive Mutants The indicated N-terminal lysine to glutamine mutations in histones H3 (A) and H4 (B) were constructed in pDP373. These were tested for their growth affect in wild-type background (M1360) and for the ability to suppress the temperature sensitivity of cdc6-4 (M1345), mcm2-1 (M1421), or cdc7-1 (M1439) mutants. Ten-fold serial dilutions of saturated cultures were spotted onto rich medium (YPD) and incubated at the indicated temperatures for 3 days (25°C) or 2 days (30°C, 35°C, and 37°C plates). Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Increased Expression of Wild-Type Histone H3 and H4 Is Lethal to cdc6-1 (A) Wild-type (M1812), orc5-1 (M1814), cdc6-1 (M1816), and mcm2-1 (M1818) strains containing a single integrated copy of a plasmid expressing HHT1 and HHF1 from the Gal1,10 promoter (Gunjan and Verreault, 2003) were spotted in 10-fold serial dilutions onto glucose plates (uninduced) and galactose plates (induced) and incubated for 3 days at 25°C. (B) The same strains were grown at 25°C in 2% raffinose medium, glucose (−) or galactose (+) was added for 3 hr, and then the level of histone H3 was determined in total cell extracts by blotting for the Flag tag on HHT1. Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 The IS Elements of ARS305 and ARS315 Occur within Stably Positioned Nucleosomes Stably positioned nucleosomes (light blue ovals) are indicated in the vicinity of ARS305, ARS315, and ARS307 (Yuan et al., 2005). A and B elements are indicated by black boxes, and IS elements by red boxes. The chromosome III coordinates are indicated above each positioned nucleosomes and for the start or stop codons of the adjacent genes (open boxes). Arrows indicate the direction of transcription. Molecular Cell 2008 30, 156-166DOI: (10.1016/j.molcel.2008.02.019) Copyright © 2008 Elsevier Inc. Terms and Conditions