Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in <em>Saccharomyces cerevisiae</em>

<div><p>The centromeric regions of all <em>Saccharomyces cerevisiae</em> chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In <em>Candida albicans</em>, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of <em>S. cerevisiae</em> origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in <em>cis</em> over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.</p> </div

A schematic diagram of chromosome XIV in wild-type (WT) and rearranged strains.

<p>(A) In the WT strain, the BglII restriction site in MET2 is located 8.5 kb to the left of ARS1410. Centromere XIV resides in its endogenous position located 6.8 kb to the left of ARS1426. (B) In the rearranged strain the endogenous centromere was replaced with a URA3 selectable marker while a functional centromere was integrated along with LEU2 (open box) into the MET2 locus such that the centromere was positioned ∼11.5 kb from ARS1410. The white and black arrowhead above each centromere indicates the direction of the centromere DNA elements CDEI, CDEII, CDEIII.</p

Models for centromere-mediated early origin activation.

<p>(A) Kinetochore/microtubule interaction orients the centromere and pericentric DNA near the microtubule organizing center (MTOC) where there is an enrichment of replication initiation factors. (B) Tension exerted by the kinetochore/microtubule interaction induces an altered chromatin structure of pericentric DNA that provides accessibility of embedded origins to initiation factors. (C) Kinetochore proteins interact directly (or indirectly) with origin initiation factors recruiting them to nearby origins. (D) The organization of pericentric DNA into the C-loop orients origins within the C-loop to the periphery of the chromatin mass increasing their accessibility to initiation factors.</p

2D gel analysis of ARS1410, ARS1426, and MET2 and met2.

<p>DNA fragments containing a functional origin are detected as a bubble arc (depicted by the bubble fragment) while fragments that are passively replicated are detected as a Y-arc (depicted as a Y shaped fragment). (A) ARS1410 and ARS1426 are functional origins in the WT and rearranged strains. (B) 2D gel analysis of the MET2 or met2 locus in the WT and the rearranged strains. WT DNA was digested with the restriction enzyme StuI giving a fragment of 3.96 kb centered on MET2 (grey arrow). Rearranged DNA was double digested with XbaI and BsrBI resulting in a 4.36 kb fragment harboring most of met2, the integrated centromere (black circle), and the LEU2 marker (white rectangle). The absence of a bubble arc when probed for the 3′ end of MET2 and met2 (hashed rectangle) indicates that an origin is not present on either DNA fragment. The centromere in the rearranged construct was detected as a pause site (black arrowhead) visualized as a dot of relatively increased intensity on the descending Y-arc.</p

Replication dynamics for chromosome XIV in WT and rearranged strains.

<p>(A) Replication kinetic profiles of chromosome XIV in WT (top) and rearranged (bottom) strains. Percent replication was monitored across chromosome XIV at 40 (magenta), 45 (orange), 55 (green), and 65 (blue) minutes following release from alpha factor arrest. When the native centromere (yellow circle) is present near ARS1426, a prominent peak is seen in the 40 and 45 minute time samples. In this strain, the peak at ARS1410 is shallow in the 40 and 45 minute samples. When the centromere is repositioned (orange circle) near ARS1410 in the rearranged strain, both the time of appearance and the prominence of the peaks at ARS1410 and ARS1426 are inverted with respect to the WT strain. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s006" target="_blank">Figures S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s007" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s001" target="_blank">Datasets S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s002" target="_blank">S2</a> for all chromosomes. (B) Z-score plots of chromosome XIV in WT (black) and rearranged (blue) strains. Replication kinetic profiles from the 40 minute sample were normalized by converting percent replication values to Z-score values (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#s4" target="_blank">Materials and Methods</a>). Genomic loci corresponding to ARS1410 and ARS1426 show significant differences in Z-scores. ARS1424 is the next closest active origin to the endogenous centromere residing ∼19 kb to the left. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s008" target="_blank">Figures S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s009" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s010" target="_blank">S6</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s003" target="_blank">Datasets S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s004" target="_blank">S4</a> for all chromosomes and the 45- and 65-minute samples.</p

Z-score and 2D gel analysis of ARS1531.

<p>(A) Z-score plot of chromosome XV in WT (black) and rearranged (blue) strains. ARS1531 displayed a difference of Z-score values at least as large as that seen for ARS1410 and ARS1426 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen-1002677-g005" target="_blank">Figure 5B</a>). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s008" target="_blank">Figure S4</a> for all chromosomes. (B) 2D gel analysis of ARS1531 in the WT (left), the rearranged strain used in microarray analysis (middle), and the rearranged strain used for prior slot blot analysis (right). DNA from all three strains was digested with NcoI and BglII to give a 3.18 kb fragment harboring ARS1531 and then subjected to 2D gel analysis. The presence of a bubble arc in the WT (black arrow) indicates that ARS1531 is a functional origin in this strain. The presence of a bubble arc in one of the two rearranged strains confirms that the absence of origin activity in rearranged A (used in microarray analysis) is not due to relocation of the centromere on chromosome XIV. Below each 2D gel image is the sequence for the WT or mutant (red) ACS.</p

The chromosomal context for <i>SUL1</i> amplification.

<p>(A) The inverted repeat sequences in <i>CTP1</i> and <i>PCA1</i> that define the breakpoints of a specific <i>SUL1</i> amplification event <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002016#pgen.1002016-Araya1" target="_blank">[7]</a>. (B) The structure of the wild type <i>SUL1</i> locus that includes the nearby origin of replication, <i>ARS228</i>. (C) The inferred structure of the head-to-head/tail-to-tail 5× <i>SUL1</i> amplification product recovered after selective growth of a haploid yeast strain in medium limiting for sulfur.</p

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast

<div><p>A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (<i>ORC1</i>, <i>ORC4</i>, <i>ORC6</i>, <i>CDC6</i>, <i>CDT1</i>, and <i>MCM5</i>). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and <i>CDC45</i>). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human <i>ORC4</i> alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (<i>orc4</i>^Y232C). We find that yeast cells with the <i>orc4</i>^Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, <i>orc4</i>^Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis.</p></div

Growth and cell cycle characterization of <i>orc4</i>^Y232C cells.

<p>(A) Growth of <i>ORC4</i> and <i>orc4</i>^Y232C cells as measured by change in optical density (OD₆₆₀) of mid-log phase cultures in synthetic complete medium at 30°C. The mutant (white circle) shows a modest growth defect with a doubling-time 18 minutes (12%) longer than wild-type cells (black circle). (B) S phase progression of <i>ORC4</i> (left) and <i>orc4</i>^Y232C (right) cells as measured by flow cytometry. Cells were synchronously released into S phase and cell samples were collected at 5-minute intervals. <i>ORC4</i> cells enter S phase ~20 minutes after release from alpha-factor, whereas <i>orc4</i>^Y232C cells entered S phase ~25 minutes after release. By 85 minutes <i>ORC4</i> cells are cycling back to begin a new cell cycle, while <i>orc4</i>^Y232C cells do not begin to cycle back until 120 minutes. The orange dotted line indicates the expected DNA content for cells that have completed replication. (C) ssDNA profiles of early origin activity in <i>ORC4</i> (blue) compared to <i>orc4</i>^Y232C (red) cells are shown for Chr VII. G1 cells were synchronously released into S phase in the presence of HU to reduce movement of replisomes away from origins of replication. The y-axis values represent the relative amounts of ssDNA calculated as the ratio of fluorescent signal of S phase sample (30 min) to G1 control. Chromosome coordinates for Chr VII are displayed along the x-axis and sites of origin initiation appear as peaks along this axis. The centromere location is represented as a yellow circle on the x-axis and verified origins of replication are marked by orange triangles. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007041#pgen.1007041.s003" target="_blank">S3 Fig</a> for the full set of profiles. (D) Scatter plot comparing the areas under the peaks of ssDNA observed in <i>ORC4</i> vs. <i>orc4</i>^Y232C cells. Origins whose activity was largely restricted to one genotype or the other are encircled by the orange ovals. (E) Sensitivity of <i>orc4</i>^Y232C cells to hydroxyurea (HU). Serial dilutions (1:3) of cells were plated on synthetic complete medium with or without HU (200 mM) and incubated at 30°C for 3 days.</p

Reduction of rDNA copy number in <i>orc4</i>^Y232C cells.

<p>(A) Flow cytometry histograms of log-phase populations of <i>ORC4</i> (blue) and <i>orc4</i>^Y232C (red) cells overlaid with one another. The mutant’s profile is shifted to the left indicating a loss (~10%) of cellular DNA content. (B) Quantitative Southern blot analysis of repetitive DNA sequences. Genomic DNA from <i>ORC4</i> and <i>orc4</i>^Y232C cells was digested with a restriction enzyme and separated by standard gel electrophoresis. The Southern blot of the gel was first probed with <i>ACT1</i> sequence (as a single copy control) and then subsequently probed with various repetitive DNA sequence including mitochondrial DNA, the native, nuclear 2-micron plasmid DNA, and rDNA. The bands above and below the major center band present when probed for 2-micron correspond to restriction fragments from the low levels of naturally occurring dimeric plasmid molecules between the A and B isomers of the plasmid. (C) Cartoon depiction of the yeast rDNA locus. The yeast rDNA locus consists of 100–200 copies of a 9.1 kb tandem repeat located on Chr XII. Each rDNA repeat encodes the template necessary to make ribosomal RNA (25S, 5.8S, 18S and 5S) and also contains an origin of replication. (D) CHEF gel analysis of Chr XII size in <i>ORC4</i> and <i>orc4</i>^Y232C cells. Left panel, ethidium bromide stained image; right panel, image of Southern blot following hybridization with a Chr XII-specific single-copy sequence. The faster migration of Chr XII in <i>orc4</i>^Y232C cells confirms the loss of chromosomal rDNA repeats from ~150 (<i>ORC4</i>) to ~30 copies (<i>orc4</i>^Y232C). (E) CHEF gel analysis of variation in rDNA copy number in six additional isolates (a-f) of <i>orc4</i>^Y232C. Left, ethidium bromide stained image; right, Southern blot hybridization for Chr XII as in (D). Samples were prepared after ~20 generations of growth following introduction of the <i>orc4</i>^Y232C mutation. All six isolates had a smaller Chr XII than <i>ORC4</i> due to loss of rDNA repeats. The rDNA copy number in the isolates ranges from ~30 copies (isolate c) to ~100 copies (isolates a, b, d, and e). (F) Long-term growth of isolate “e” shows rDNA copy number stabilizes at ~30 copies. After confirming isolate “e” to have the mutant <i>orc4</i>^Y232C allele, cells were continuously passaged for ~100 generations in batch culture. Cells were allowed to grow to saturation after each passage. For each sample cells were collected and whole chromosomes were separated by CHEF gel electrophoresis (ethidium bromide stained image on left, Southern hybridization for Chr XII). By ~80 generations (*) the rDNA copy number stabilized at ~30 copies in the population.</p