Altered cell cycle distribution and replication fork progression in ORC1 knockdown cells.

<p>(A) DNA samples from log phase cultures were subjected to neutral-neutral 2D gel analysis following digestion with <i>HindIII</i> and enrichment for RIs on BND cellulose. Left panels: blots were probed with the rDNA 5′ NTS probe (wild type (WT) and ORC1 knockdown (ORC1-KD) strains. Right panel, schematic of the palindromic <i>HindIII</i> fragment spanning the two inverted copies of the 5′ NTS, promoter (pro) and replication origins (D1 and D2). (B) Representative images for DNA fibers sequentially labeled with IdU and CldU. Inter-origin distance and fork velocity were measured in log phase CU428 and ORC1-KD cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004875#s4" target="_blank">Materials and Methods</a> for details).</p

ORC1 depletion induces slow cell cycle progression.

<p>(A) Western blot analysis of whole cell lysates (WL), NP-40-extractable soluble fractions (S), and nuclear chromatin-bound pellet fractions (P). Samples were prepared from log phase wild type (CU428) and ORC1 knockdown (ORC1-KD) cells, and immunoblotted with rabbit polyclonal anti-Orc1p, anti-Orc2p, anti-Mcm6p, and anti-acetyl Histone H3 antibodies. Each lane corresponds to proteins derived from 10 µl of cultured cells at density of 2 × 10⁵ cells/ml. Membranes were stained with Ponceau S to visualize total protein loaded in each lane prior to antibody probing. (B) Western blot analysis of chromatin bound pre-RC components in wild type and ORC1 knockdown strains. A Lowry assay was performed to assure that equivalent amounts of protein (20 µg) were loaded in each lane. Due to the different sizes of target proteins, a single membrane was cut into pieces to probe for each target protein. (C) Cell cycle progression of CU428 and ORC1-KD cells as measured by flow cytometry. 0 min corresponds to G1 phase cells isolated by elutriated centrifugation. (D) Flow cytometry analysis of asynchronous, log phase wild type (CU428) and ORC1 knockdown (ORC1-KD) cells. Vegetative growing cell cultures were harvested at late log phase (cell density: 2.5×10⁵ cells/ml).</p

Abrogated intra-S phase checkpoint response in ORC1 knockdown cells.

<p>(A) Elutriated G1 phase wild type (CU428) and ORC1 knockdown (ORC1-KD) cells were treated with 20 mM HU and samples were collected at the indicated intervals for flow cytometry analysis. (B) G1 synchronized cells were released into fresh medium containing 20 mM HU or 0.06% MMS +/− 1 mM caffeine (1 mM) for 4 h. Whole cell lysates were subjected to western blot analysis of Rad51p. (C) G1 synchronized cells were incubated in medium containing 20 mM HU. Whole cell lysates were prepared at timed intervals and subjected to western blot analysis with anti-Rad51 antibody. (D) G1 synchronized cells were incubated in the presence of HU (1–20 mM) for 4 h and subjected to western blot analysis. (E) Alkaline gel electrophoresis of nascent DNA strands accumulated under HU treatment. G1 synchronized cells were cultured in 20 mM HU and genomic DNA was isolated at indicated time points. RIs were released under alkaline condition and resolved in a 1% alkaline agarose gel. RIs from the rDNA 5′ NTS origin region were visualized by Southern blot analysis.</p

Endoreplication and rDNA amplification during Tetrahymena development.

<p>(A) Flow cytometry analysis of matings between wild type and ORC1-KD strains. Nuclei were isolated and stained with propidium iodide for flow cytometric analysis. Wild type strains: CU427, CU428, SB1934. SB1934 is a heterokaryon, with B rDNA in the macronucleus and C3 rDNA in the micronucleus (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004875#pgen.1004875.s005" target="_blank">S1 Table</a>). OM, old parental macronucleus, which is degraded in conjugants. (B) Cytological examination of crosses WT X WT (SB1934 and CU428), and WT X mutant (SB1934 and ORC1-KD) with the DNA staining dye, DAPI. Starved mating cultures were re-fed at 24 h. Three representative images are shown for each time point. (C) Southern blot analysis of C3 rDNA gene amplification during development. Mating between wild type SB1934 or CU428 strains with one another (WT x WT) or with the ORC1-KD strain (WT X mutant) were performed and cells were collected at the indicated time points. DNA was digested with <i>BamHI</i> and probed with an rDNA 3′ NTS probe to distinguish macronuclear B (4.0 kb) and C3 (2.5 kb) rDNA alleles.</p

DNA fiber analysis of mock-treated cells and cells recovering from HU-induced replication stress.

<p>An asynchronous cell population was used to measure inter-origin distances and fork rates in mock-treated cells by sequential labeling with IdU and CldU for 10 min each (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#sec013" target="_blank">Materials and Methods</a>). HU-treated cells were synchronized at the G1/S border by starvation and re-feeding, and released into 20 mM HU for 4 h. Cells were washed free of HU, and incubated for 60 min prior to the sequential addition of IdU and CldU for 10 min each. (A) Representative DNA fibers images. (B) Compilation of DNA fiber image data and statistical analysis. Inter-origin distance was defined as the distance between the centers of two red segments in either green-red-green-red-green or green-red-gap-red-green tracks. Fork velocity was determined by measuring the length of the green segment in red-green tracks or the red segments in green-red-gap-red-green tracks. GraphPad Prism software was used to analyze the statistical significance, and the p-values shown in figures were determined by two-tailed unpaired t-test.</p

Two-dimensional gel electrophoresis analysis of rDNA replication intermediates following removal of HU.

<p>Cells were synchronized at the G1/S border by starvation and cultured in growth media in the absence or presence of 20 mM HU. DNA was prepared from mock-treated starved cells, mock-treated S phase cells, and HU-treated cells at defined intervals following HU removal. (A) Upper diagram: schematic of the 21 kb rDNA minichromosome and location of relevant restriction sites and probes for Southern blot analysis. Macronuclear rDNA minichromosomes contain two copies of the rRNA coding region and adjacent 5' and 3 'non-transcribed spacer (NTS) regions in an inverted orientation. The 35S rRNA precursor encodes the 17S, 5.8S and 26S rRNAs (grey areas- mature RNA coding regions, black and white stippled areas- processed RNA precursor regions, hatched area- self-splicing 26S rRNA intron). Telomeric DNA repeats (vertical hashes) are present at chromosome termini. The positions of four probes for N/N and N/A 2D gel analysis are shown. Expanded view of the 1.9 kb 5 NTS. Thick arrow- rRNA promoter; grey ovals- position of phase nucleosomes in vegetative rDNA minichromosomes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#pgen.1005405.ref060" target="_blank">60</a>], black boxes- type 1 repeats. Domains 1 and 2 (thin arrows) are 430 bp imperfect tandem repeats with 230 bp nuclease hypersensitive regions Lower diagram: schematic of typical RI patterns detected by N/N 2D gel electrophoresis. Simple Y arc (arrow): passive replication of the probed DNA fragment interval due to initiation elsewhere in the chromosome. Bubble arc (arrowhead): initiation at a central position in the probed DNA fragment. Bubble-to-Y arc: initiation at an asymmetric position in the examined fragment (low MW RIs: bubble arc (arrowhead), high MW RIs: Y arc). Composite: active (complete bubble arc, arrowhead) and passive (complete Y arc, arrow) replication within the probed DNA fragment. X and Double Y: the X spike (arrowhead) is generated from branch migration recombinant intermediates, and the double Y (open arrow) is generated by two converging replication forks. (B) RI patterns detected with the 5’ NTS probe 1 on <i>HindIII</i> digested DNA from mock treated quiescent (starvation) and replicating cell populations (S phase). (C) 5’ NTS analysis on DNA from HU-treated cells 0–120 min after HU removal (arrow: simple Y arc, passive replication; arrowhead: X-spike recombination intermediates). See flow cytometry profiles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#pgen.1005405.g005" target="_blank">Fig 5A</a>) and western blots (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#pgen.1005405.g005" target="_blank">Fig 5B</a>) for cell cycle progression and abundance of Orc1p and Mcm6p, respectively. (D) Two-dimensional N-N gel analysis of the 5.5 kb rRNA coding region <i>ClaI</i> fragment (position 2168–7629, probe 2). (E) Two dimensional neutral-alkaline (N/A) analysis of RIs derived from the rRNA coding region <i>ClaI</i> fragment. Probe 3 spans nucleotides 2169 to 3670, and probe 4 spans nucleotides 5214–6676. Schematic of nascent-strand RIs resolved by N/A 2D gel electrophoresis. The 1n spot corresponds to non-replicating DNA. The vertical smear is derived from nicked, non-replicating DNA, and the horizontal smear represents the parental strand in RIs of different length. The diagonal arc corresponds to nascent-strand replication intermediates that are liberated from the parental strand by alkali denaturation prior to electrophoresis in the second dimension.</p

Characterization of HU and MMS induced checkpoint responses.

<p>(A) Western blot analysis of Rad51p for asynchronous log phase cultures (left panel) or elutriated G1 phase cells (right panel) treated for 4 h with 20 mM HU, 0.06% MMS, 1 mM caffeine, or in combination. Equivalent amounts of protein were loaded in each lane. Ponceau S staining was used to verify protein transfer and serve as a crude validation for equal protein loading prior to antibody probing. (B) G1/S and intra-S phase checkpoint analysis. Cells were synchronized at the G1/S border by centrifugal elutriation. 20 mM HU or 0.06% MMS were added immediately (G1 phase addition, left panel) or 1 h later (mid-S phase addition, right panel). Western blot analysis was performed with antibodies specific directed against Rad51p and Mcm6p. The zero hour time point (right panel) corresponds to freshly elutriated G1 cells for both the G1 (left panel) and mid-S phase (right panel) treatment regimens. (C) Flow cytometry analysis of cell populations analyzed in B. The vertical red line demarcates the peak at the time of drug addition. The arrow (4 h post drug addition) in cultures treated with HU or MMS during S phase demarcates the newly formed G1-like peak. (D) Flow cytometry profiling during an extended HU treatment of S-phase cells. Left panel: mock-treated cells. Right panel: HU-treated cultures in which elutriated G1 cells were grown for 1 h (to early/mid S phase) prior to addition of 20 mM HU for 0–6 h. (E) Data from HU-treated mid-S phase cells. Control flow cytometry profiles for mock-treated cells in S phase (red line, 1 h after G1 isolation) and G1+ G2 phase (green line, 2 h after G1 isolation). Treated cells cultured with HU for 4 h (blue line, HU treatment beginning 1 h after elutriation). Red arrows: newly formed peak at the G1 position in HU-treated cells (blue profile). Red arrowhead: right shoulder for the cell population at time of HU addition (red profile) and 4 h late (blue profile).</p

Effect of HU and MMS on pre-RC proteins in G1 treated cultures.

<p>HU (20 mM) or MMS (0.06%) were added immediately to an elutriated G1 phase population and cells were cultured for 4 h. (A) Flow cytometry analysis at 1 h intervals. (B) Western blot analysis with Rad51p and Mcm6p antibodies. Twenty micrograms of protein was loaded in each lane, as determined by Lowry protein assay, and Ponceau S staining was used to assess protein transfer prior to antibody probing. (C) Western blot analysis with Rad51p, Orc1p and acetylated histone H3 antibodies. Cells were synchronized by starvation and re-feeding, and cultured for 4 h +/- HU. (D) Western blot analysis of cells treated with 20 mM HU in the presence or absence of ATR and histone deacetylase inhibitors (1 mM caffeine and 50 mM sodium butyrate, respectively). For panel D, cells were synchronized by starvation and re-feeding, and subsequently cultured for 4 h. Western blot analysis with antibodies directed against Orc1p, Mcm6p and Rad51p.</p

Effect of HU on DNA replication and acetylation of histone H3.

<p>(A) DNA fiber analysis was performed in the presence or absence of HU to assess nascent strand synthesis and stability. Cells were synchronized at the G1/S border by starvation and re-feeding, and cultured in the presence of 20 mM HU and 400 μM IdU for 4 h, or 20 mM HU for 3 h, then 20 mM HU plus 400 μM IdU for 1 h. HU and IdU were removed, and cells were further cultured for 90 min in media containing 100 μM CldU. DNA fibers were visualized as described in Materials and Methods (IdU, red; CldU, green). As a control, an untreated log phase culture was sequentially pulse labeled for 20 min in media containing IdU and CldU. (B) Mid-S phase cells were pulse labeled with 100 μM CldU for 10 min prior to the addition of HU, and DNA fibers were prepared 0, 1 and 4 h after HU treatment. The length of individual labeled DNA tracts was plotted and the median tract length was calculated as subjected to statistical analysis. (C) Western blot analysis with phospho-gamma H2A.X and acetylated histone H3 antibodies. Synchronized starved/re-fed cells were treated for 4 h with the indicated drug or drug combination (20 mM HU, 1mM caffeine, 50 mM sodium butyrate) and cells were lysed in SDS loading buffer. Twenty micrograms of protein was loaded in each lane. Ponceau S staining was used to verify protein transfer and served as a crude validation for equal protein loading prior to antibody probing. (D) Flow cytometry profiles of elutriated cells treated with 20 mM HU or 20 mM HU and 50 mM sodium butyrate for 1–4 h.</p

Physical characterization of cells in mock, 20 mM HU and 0.06% MMS treated cultures.

<p>A G1 cell population was obtained by centrifugal elutriation and propagated for 1 h (mid-S phase) prior to the addition of HU or MMS. (A) Cell density analysis by direct counting on a hemocytometer). (B) Visualization of the cytokinetic furrow (a marker for cell division) in fixed DAPI-stained cells. Approximately 40% of mock-treated cells generated a cytokinetic furrow 2 h after G1 phase isolation (1 h after HU or MMS addition in treated samples). T = 4 h, mock versus HU: (student T test) two-paired P value 0.0002); T = 4 h, mock versus MMS: (student T test) two-paired P value <0.0001). (C) Plot of DNA content (PI, propidium iodide) in mock and HU-treated cells as a function of exposure time. Elutriated G1 phase cells were treated with 20 mM HU beginning 1 h after isolation (vertical arrow, mid-S phase addition) and samples were collected every 30 min for 4.5 h. 30,000 cells were scored at each time point. (D) Analysis of flow cytometry side scatter (SSC) in mock and HU-treated cells. HU was added during S phase and samples were collected at 1 h intervals and subjected to statistical analysis. The results of three independent experiments (n = 3) are compiled. (E) Visual representation of flow cytometry side scatter (SSC) and forward scatter (FSC) in cells synchronized by centrifugal elutriation. Controls: mock-treated G1 cells (T = 0 h after elutriation), mock-treated S phase cells (T = 1 h after elutriation), and mock-treated cells 6 h after elutriation. Experimental samples: 6 h HU exposure beginning in G1 phase (0 h after elutriation; (HU/G1, 6 h), or in S phase (1 h after elutriation (HU/S, 6 h). The higher complexity seen in HU-treated cells was not observed at any stage of the cell cycle in untreated controls (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005405#pgen.1005405.s003" target="_blank">S3 Fig</a>). (F) Left panel: the total cell area for 100 microscopic cell images was measured in mock S phase and HU-treated cells. The average area was determined and subjected to statistical analysis. A 30% increase in cell size was observed in HU-treated cells (T = 8 h HU versus mock S phase). Student T test two-paired P value <0.0001 (highly significant). Right panel: representative micrograph illustrating the size difference between mock-treated S phase cells and S phase cells arrested with HU for 8 h. (G) Total macronuclear area for 100 microscopic cell images was measured in mock and HU-treated cells. The average area was determined and subjected to statistical analysis. A 20% decrease in the size of the macronucleus was observed in HU-treated cells (T = 6 h HU versus mock G1 phase cells). Student T test two-paired P value <0.1 (significant). (H) Production of macronuclear extrusion bodies. DAPI staining was used to identify cells with DNA masses that were not associated with the micro- or macronucleus (macronuclear extrusion bodies). T = 4 h, mock versus HU. Student T test, two-paired P value: 0.2249 (not significant).</p