1. Nucleosomes Positioned by ORC Facilitate the Initiation of DNA Replication 
J.Russell Lipford, Stephen P Bell 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1
Interaction of ORC and Abf1p with ARS1 Chromatin Assembled In Vitro
(A) Mnase digestion of ARS1 chromatin templates assembled in vitro in the absence or presence of purified ORC. ORC was added as indicated, and the resulting templates were treated with decreasing concentrations of Mnase. The resulting DNA fragments were analyzed by agarose gel electrophoresis. DNA markers are indicated at left.
(B) Primer extension Dnase I footprinting analysis. ORC and Abf1p were added as indicated to the ARS1 chromatin assembly reaction. Vertical lines denote the ORC and Abf1p binding sites.
(C) IEL analysis. Naked ARS1 templates (lanes 1 and 2) or templates assembled into chromatin (lanes 3–10) were digested with Mnase and then analyzed by indirect end labeling (IEL). ORC and Abf1p were included in the chromatin assembly reactions as indicated. Heavy vertical lines indicate the region corresponding to ARS1. Asterisks and crosses denote sites of Mnase digestion that are altered by the inclusion of ORC and Abf1p, respectively. Sizes of nucleosomal DNA markers are shown to the left. A model for the nucleosomal structure of ARS1 chromatin-assembled templates is shown to the right. The vertical line with an asterisk indicates location of the probe used in the IEL analysis. Distance from the IEL probe to ARS1 is also indicated.
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )

3. Figure 2
Determinants of the Nucleosomal Structure of Replication Origins In Vivo
IEL analysis of the ARS1 region of chromatin templates recovered from yeast. Flanking diagrams show models for the nucleosomal structure of each of the strains.
(A) Comparison of a wild-type strain (WT) with a strain harboring a linker substitution mutation in the ORC binding site at ARS1 (A−). Locations of the elements of ARS1 are indicated as A, B1, B2, and B3. Horizontal lines represent sites interpreted to correspond to the linker regions between nucleosomes.
(B) Comparison of a WT strain with a strain harboring a linker substitution in the B2 element of ARS1 (B2−). Asterisks indicate an Mnase digestion site that is altered in the mutant strain. Sizes of nucleosomal DNA markers are shown to the left and also apply to (A).
(C) Comparison of a WT strain with a strain harboring a linker substitution in the Abf1p binding site at ARS1 (B3−). IEL analysis was performed for the opposite strand as that in (A) and (B). Horizontal lines indicate as in (A).
(D) Comparison of a WT strain with a strain having a point mutation in the ORC binding site of ARS307 (A−). Sizes of nucleosomal DNA size markers and locations of origin elements A, B1, and B2 are shown.
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )

4. Figure 3
The Nucleosomal Configuration of the In Vitro ARS1 Chromatin Templates Mimics that of ARS1 In Vivo
(A) Comparison of IEL analyses of chromatin templates recovered from wild-type strains with those assembled in vitro that included ORC and Abf1p. Asterisks indicate sites of Mnase digestion that are interpreted to represent linker regions between nucleosomes. Sites dependent upon ORC and Abf1p are denoted and ARS1 sequence elements are also indicated.
(B) As in (A), except the opposite DNA strand was analyzed.
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )

5. Figure 4
Alteration of the Nucleosome(s) Adjacent to ORC Disrupts Plasmid Maintenance
(A) Schematics of the proposed nucleosomal structure of plasmid constructs containing either the wild-type ARS1 sequence (WT) or variants that contain binding sites for Abf1p (B3x2) or lac repressor (LacO), 70 bp away from the ORC binding site.
(B) In vivo IEL analysis of indicated plasmid constructs. Lac repressor was expressed as indicated (na, not applicable; +, lac repressor expressed; −, not expressed). Bullets indicate Mnase digestion sites interpreted to correspond to linker regions between nucleosomes. Angled lines indicate shift of the Mnase digestion pattern. ARS1 elements and sites for Abf1p and Lac repressor are also denoted.
(C) Plasmid stability assays were performed for indicated plasmids. Standard deviations calculated from at least four independent transformations.
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )

6. Figure 5
Alteration of the Nucleosomes Adjacent to a Chromosomal Origin Disrupts Initiation
(A) IEL analysis (as in Figure 2) of chromosomal ARS1 derivatives. IEL analyses were performed in wild-type strains (WT) or strains harboring the following mutations at the chromosomal ARS1 locus: a linker substitution in the A element (A−); a linker substitution in the B2 element (B2−); an additional Abf1p binding site adjacent to the A element (B3x2); and a combination of the B2− and B3x2 alterations (BB).
(B) Analysis of replication intermediates derived from the chromosomal ARS1 derivatives by two-dimensional gel electrophoresis. Strains used are the same as in (A). Y arcs and bubble arcs are indicated.
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )

7. Figure 6 Chromatin Perturbation Affects Assembly of the Pre-RC
(A) Chromatin immunoprecipitation analysis of ARS1, ARS305, and URA3 loci using polyclonal antibodies to ORC. Analyses were performed in wild-type strains (WT) or strains harboring the following mutations at the chromosomal ARS1 locus: a linker substitution in the A element (A−); a linker substitution in the B1 element (B1−); a linker substitution in the B2 element (B2−); an additional Abf1p binding site adjacent to the A element (B3x2); and a combination of the B2− and B3x2 alterations (BB). Analysis of immunoprecipitated and total input DNA is shown.
(B) Chromatin immunoprecipitation analysis of ARS1, ARS305, and URA3 loci using anti-HA antibodies to precipitate Mcm3-HA. The strains used are denoted as in (A).
Molecular Cell 2001 7, 21-30DOI: ( /S (01) )