1. The Effect of Replication Initiation on Gene Amplification in the rDNA and Its Relationship to Aging 
Austen R.D. Ganley, Satoru Ide, Kimiko Saka, Takehiko Kobayashi 
Molecular Cell 
Volume 35, Issue 5, Pages (September 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 rDNA Structure and the rDNA Amplification Model
(A) Structure of the rDNA in S. cerevisiae. There are ∼150 tandem copies of rDNA on chr XII. Each repeat unit consists of the 35S and 5S rRNA genes separated by one IGS. The IGS contains a variety of functional elements, such as promoters, terminators, an origin of replication, and an RFB site.
(B) The RFB-dependent rDNA amplification model. Three rDNA units are represented and replication starts from the rARS in unit (2). A DSB forms at the RFB site through Fob1p binding. Repair of this DSB can lead to either no copy number change through equal sister chromatid recombination (ESCR) or to copy number change through USCR. Thus, USCR can potentially occur with either the replicated region (as shown) or the unreplicated region on the other side of the RFB site. The choice between ESCR and USCR is dependent on the transcriptional status of the noncoding promoter, E-pro, that affects cohesin association. Brackets indicate a blow-up of unit (2) where the DSB has been induced. See text for further details.
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3. Figure 2 Role of the Replication Origin in rDNA Amplification
(A) Modifications of the rARS in the two-copy rDNA strain. The structure of the two-copy strain is shown above, and below are the rARS modifications. We modified rARS(1), whereas the other rARS, rARS(2), does not participate in rDNA amplification. Black boxes in the rARS correspond to the ARS consensus sequences. See text for details. N and B are NheI and BglII restriction sites, respectively.
(B) A functional rARS is required for rDNA amplification. FOB1 or empty vector (fob1) were introduced into rARSΔ2−copy. After ∼40 generations, the DNA was isolated and analyzed by CHEF. Chr XII was identified by probing blots of these gels with an rDNA fragment. Experiments were performed four times.
(C) (I) ARS activity of modified rARS sequences. Stabilities of plasmids carrying the modified rARS fragments used to make the modified rARS strains in (A) were measured. Results are plotted as percent of colonies able to grow on selective medium after growth on nonselective medium, and standard deviations are shown. (II) rDNA amplification was induced in rARSΔ-32-copy, WT2-copy, and ARS1-R2-copy by introduction of FOB1. After ∼40 generations, the DNA was isolated and analyzed by CHEF. The electrophoresis conditions used here result in DNA longer than ∼1.6 Mb stacking together to form a “band” in this region of the gel. Chr IV (upper) and chr VII and XV (two chromosomes; lower) are visible in the EtBr-stained gel, whereas chr XII is difficult to see because it is so smeared. The position of chr VII/XV is similar to that of the two-copy rDNA chr XII. Experiments were performed in duplicate. Size markers (M) are H. wingei chromosomes (Bio-Rad).
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4. Figure 3
rDNA Amplification Rate Is Partially Dependent on rARS Activity
rARS-modified strains were grown for differing periods after FOB1introduction and DNA was isolated and analyzed by CHEF as in Figure 2 at specific time points (see Experimental Procedures for details).
(A) After ∼120 generations rARSΔ-3 showed reduced rDNA amplification compared to wild-type. However, rDNA amplification was similar in the ARS1-R and wild-type strains.
(B) rARS modified strains with a chromosomal FOB1 gene (see Experimental Procedures) after ∼200–500 generations. All strains, including rARSΔ-3, reached wild-type copy number. Experiments were performed in duplicate.
(C) Replication initiation activities were measured using 2D gel electrophoresis. A schematic representation of the various arcs and spots is shown in the right panel. Bubble arc signals were normalized to the Y arc signal, and relative initiation efficiencies (to wild-type) are shown below.
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5. Figure 4
Life Span Correlates with rDNA Stability Rather Than ERC Level
(A) Detection of ERC in the rARS modified strains. Undigested DNA isolated from WTamp, ARS1-Ramp, and rARSΔ-3amp was separated by gel electrophoresis, and the rDNA was detected by probing with an rDNA fragment. Bands corresponding to genomic rDNA and the different ERC forms are indicated.
(B) The intensities of monomer and dimer ERC bands from (A) were quantified and normalized to genomic rDNA. Values are relative to WTamp. This was performed in triplicate, and standard deviations are shown.
(C) Replicative life span of the rARS modified strains. Life spans were determined by scoring the number of daughter cells produced by each mother cell before cessation of all division and are shown as mortality curves. Average life spans were: WTamp = 22.3; ARS1-Ramp = 18.0; rARSΔ-3amp = The results are presented in Table 1 and are shown as scatter plots in Figure S4.
(D) Stability of the rDNA as determined by marker loss assay. Rate of loss of the marker gene (ADE2) by unequal recombination in the rDNA repeats is plotted, normalized to wild-type. This was performed in triplicate, and standard deviations are shown.
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6. Figure 5 rDNA Stability in Mother and Daughter Cells
(A) Mother and daughter cells were separated by size using an elutriator, and bud scars were observed using calcofluor staining. In the mother fraction, most cells have multiple scars, while scars are not observed in the daughter fraction.
(B) Chromosomes were analyzed by CHEF. Chromosomes undergoing recombination cannot migrate due to the presence of branched intermediates and therefore are stuck in the well. Chr XII (rDNA) and chr II (control) were analyzed. For each chromosome, the left panel is the EtBr-stained gel and the right panel is a Southern blot of this gel with the appropriate chromosomal probe. Results are shown for mother cells (M) and their daughter cells (D).
(C) Quantification of the results from (B). The intensity of chromosomal signal from the Southern blot that does not migrate into the gel is plotted relative to total intensity of chromosome bands in the gel and well. Aging mutants (fob1, sir2, and fob1 sir2) were analyzed similarly (Figure S6). Standard deviations are shown.
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7. Figure 6 rDNA Instability and Aging
(A) USCR is affected by replication bubble size. (I) In wild-type, the replication (rep) bubble size is large (from the fusion of several smaller bubbles) and the broken end at the RFB (shown by red DSB) tends to recombine with the sister chromatid either equally (orange arrowhead) or unequally within the bubble (green arrow). This results in no change or an increase in rDNA copy number, respectively. (II) In the rARSΔ-3 strain, bubble size is small and the broken end tends to recombine with the sister chromatid either equally (orange arrow) or unequally with the unreplicated (nonbubble) region. USCR with the unreplicated region results in loss of rDNA copy number (blue arrows) or no change in rDNA copy number (red arrows), depending on which side of the blocked RFB site the broken end recombines with. (III) Expected outcomes from the recombination events diagrammed in (II). The structure expected from the thick red arrow (IIIa) and the structure expected from the thick blue arrow (IIIb). The outcomes of recombination events in (I) are shown in Figure 1B.
(B) Model for mother-specific rDNA instability. During cell division, damaged dysfunctional nucleolar proteins (dark gray) stay in the mother cell and nondamaged functional proteins (light gray) move to the daughter cell as the rDNA segregates. Damaged nucleolar proteins result in rDNA instability in the mother, inducing cellular senescence (orange line), while functional repair enzymes recover rDNA stability in the daughter, resulting in rejuvenation. ERC accumulation also occurs in the mother, increasing rDNA instability. In this model, rDNA instability gradually reduces the quality and quantity of ribosomes, decreasing cellular function, and finally stopping cell division.
Molecular Cell  , DOI: ( /j.molcel )
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