The Orc1BAH domain contributed differentially to ORC's association with ARSs. ChIP-on-chip experiments were performed with anti-Orc1 antibodies from ORC1 and orc1bahΔ cells. The chips used were custom-designed high-density tiled arrays from Nimblegen (Shor et al. 2009). (A) Overlap between the peaks identified in the ORC1 array with a P-value cutoff <10⁻³⁰ in this study with the likely and confirmed ARSs listed in OriDB (568 sites) (Nieduszynski et al. 2007) (top), and the ORC and ORC/Mcm2 peaks identified by Aparicio and colleagues (Xu et al. 2006) (bottom). (B) Graph of number of peaks identified in the ORC1 wild-type array (Y-axis) against their orc1bahΔ/ORC1 ratio (X-axis). (C) Peaks from microarray experiments for representative ARSs.

The orc1bahΔ and orc2-1 mutations affected different features of origin recognition by ORC. (A) For all the likely and confirmed ARSs identified in the ORC1 array, their orc1bahΔ/ORC1 peak size ratios were determined (Y-axis) (this study) and plotted against their corresponding orc2-1/ORC2 peak size ratio (Shor et al. 2009). (B) Representative tetrads from crosses between orc2-1 and orc1bahΔ cells and orc2-1 and sir1Δ cells. Viable haploid cells containing both the orc2-1 (measured by temperature-sensitive growth) and orc1bahΔ (measured by PCR) alleles were not recovered from 10–20 tetrads examined from three independent crosses. Tetrads are shown from a cross between CFY1108 (MATa orc1bahΔ) and CFY274 (MATα orc2-1), and between CFY274 (MATα orc2-1) and CFY430 (MATa sir1Δ∷LEU2). The orc2-1 allele was identified by temperature-sensitive growth and the orc1bahΔ allele was identified by PCR. The sir1Δ∷LEU2 allele was identified by growth on minimal medium lacking leucine.

Difference between the core ORC-binding site of orc1bahΔ-sensitive and orc1bahΔ-resistant origins. (A) The consensus sites for the predicted ORC site and surrounding sequences were determined for the 35 orc1bahΔ-sensitive, the 29 orc1bahΔ-resistant, and all 296 of the confirmed ARSs (as listed in OriDB) using WebLogo (Crooks et al. 2004). The 15-bp core ACS (A-element) and the 3-nucleotide B1-element are indicated with a line below the consensus site shown for all 296 ARSs (Xu et al. 2006; Chang et al. 2008). (B) Plasmid loss rate data from ARS1323 (pCF1903), ARS804 (pCF1911) (as shown in Fig. 5B), and the two different ARS–ORC-binding site swaps (ARS1323.804A–B1 [pCF1982], and ARS804.1323A–B1 [pCF1981]).

The Orc1BAH domain contributed to ORC's association with chromatin in yeast. (A) Outline of protocol used to compare immunopurified ORC from chromatin extracts prepared from ORC1 wild-type or orc1bahΔ mutant cells. See Supplemental Figure S1 for more detail. (B) The top panel on the left reports average iTRAQ ratios calculated for each ORC subunit except Orc1. (The Applied Biosystems algorithm could not determine the average iTRAQ ratio for the Orc1BAH domain and the remaining portion of Orc1, as it uses wild-type S. cerevisiae protein database as reference.) The top panel on the right reports average iTRAQ ratios calculated for each of the core histones and yeast histone H1. The average iTRAQ orc1bahΔ/ORC1 ratio for individual ORC subunits (average of all of the orc1bahΔ/ORC iTRAQ peptide ratios that comprised each polypeptide, determined by an Applied Biosystems algorithm) was close to 1.0, indicating that an anti-Orc1 IP recovered similar levels of ORC from wild-type ORC1 and orc1bahΔ mutant cells. In contrast, the average iTRAQ ratio for each of the core histones was ≤0.4, indicating that histones were recovered less efficiently from orc1bahΔ mutant compared with ORC1 cells. The bottom panel is a protein immunoblot of Orc1, Orc3, and Orc4 subunits from ORC1 wild-type and orc1bahΔ mutant cell extracts. A₆₀₀ cell equivalents of 0.25 (lanes 1,3) and 0.5 (lanes 2,4) were loaded. (C) Crude extracts from ORC1 wild-type or orc1bahΔ mutant cells (lanes 1–4, total) were fractionated by centrifugation into soluble (lanes 5–8, soluble) and chromatin-containing pellet (lanes 9–12, pellet) fractions. A₆₀₀ cell equivalents of 0.38 O.D. and 0.75 O.D. of total extracts and soluble and pellet fractions were analyzed by protein immunoblotting for the indicated proteins. Orc1 from wild-type cells produced three detectable protein fragments in these experiments, only one of which corresponded to predicted full-length Orc1 protein, presumably because of Orc1 protein degradation. The transcription factor Fkh1 was also analyzed in this experiment, with polyclonal antibodies against Fkh1 (Casey et al. 2008). The cytosolic chaperone Sis1 was analyzed with polyclonal antibodies against Sis1 (Yan and Craig 1999).

Mutation of Orc1BAH specifically affects ORC and MCM association with an orc1bahΔ-sensitive ARS. qPCR of ChIP experiments was used to analyze ORC and MCM association with ARS1323 and ARS804. (A) Diagram of ARS1323 and ARS804. Numbers below the gray lines refer to the ∼100-bp regions detected by primers described in Supplemental Table S4. (B) Orc1-directed ChIP of these ARSs show that either deletion or point mutation (A2P or E95K) of Orc1BAH reduced ORC association with the orc1bahΔ-sensitive ARS1323 but not the orc1bahΔ-resistant ARS804. (C) MCM-directed ChIPs show that deletion of Orc1BAH reduced MCM association with a region near ARS1323 (region 4) but not ARS804.

The Orc1BAH domain modulated replication initiation. (A) Two-dimensional origin mapping on the indicated chromosomal origins in ORC1 (CFY145) and orc1bahΔ (CFY1108) cells. The orc1bahΔ/ORC1 peak size ratio as determined from the ChIP-on-chip data is indicated parenthetically next to the ARS name. The arrows indicate examples in which origin bubble intermediates show increased heterogeneity in orc1bahΔ cells. Two different exposures of film are shown for the 2D origin mapping experiments for ARS1211 and ARS432.5 in orc1bahΔ cells. Details about restriction fragments and radioactive probes used for these experiments are included in Supplemental Table S3. (B) Results from plasmid loss rate assays for the indicated strains and various ARS-containing plasmids: ORC1 (CFY145), orc1bahΔ (CFY1108), sir1Δ (CFY345), nat1Δ (CFY2922), and orc1bahΔ nat1Δ (CFY3086).

Differences in nucleosome positioning surrounding orc1bahΔ-sensitive and orc1bahΔ-resistant origins. (A) The average size in base pairs of the nucleosome-depleted regions (NDRs; also called NFRs) containing the orc1bahΔ-sensitive (S), orc1bahΔ-resistant (R), and all ARSs identified in our ChIP-on-chip and predicted to exist within an NFR was determined. The data are presented as a box-and-whiskers plot (Y-axis indicates size of NFR in base pairs) and the P-value confidence value for comparisons of the means of the S and R NFRs are indicated. The mean NFR for each class of ARS is indicated below the X-axis. The asterisk (*) indicates outlier ARSs in each category. Two different data sets were used independently in these analyses, from Lee et al. (2007) and Mavrich et al. (2008). (B) The average nucleosome signal (Y-axis) was plotted for each nucleotide position relative to the T-rich strand of the ACS (with the 0 position of the ACS as indicated in Fig. 6A) for orc1bahΔ-sensitive (S), orc1bahΔ-resistant (R), and all other (A) ARSs. The nucleosome positioning data set was from Lee et al. (2007). Shifts in the −1 and −2 nucleosomes and the linker space between the −3 and −2 nucleosomes for the S class are indicated with arrows. This analysis was repeated with a more recently generated whole-genome nucleosome positioning data set with similar results (Supplemental Fig. S4).