Figure 1.
ORC1 depletion induces slow cell cycle progression.
(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).
Figure 2.
Abrogated intra-S phase checkpoint response in ORC1 knockdown cells.
(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.
Figure 3.
Altered cell cycle distribution and replication fork progression in ORC1 knockdown cells.
(A) DNA samples from log phase cultures were subjected to neutral-neutral 2D gel analysis following digestion with HindIII 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 HindIII 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 Materials and Methods for details).
Figure 4.
Micronuclear genome instability in ORC1 knockdown cells.
(A) ORC1 knockdown (ORC1-KD) cells were propagated following the establishment of clonal lines. Genomic DNA was isolated at 120 and 250 fissions and subjected to PCR amplification with the primer sets that span sites for chromosome breakage sequence (CBS)-mediated chromosome fragmentation in the developing macronucleus. PCR primers derived from the right (R) and left (L) arms of all five micronuclear chromosomes were tested. 1–10, clonal ORC1-KD; W, wild type strain CU427; (−), PCR reactions in the absence of template DNA. (B) Cytological examination of crosses between wild type strains (CU427 X CU428), or CU427 X ORC1-KD. Nuclei were visualized with the DNA staining dye DAPI. Cartoons depict the progression of wild type mating cells during development. 3 h: micronuclear elongation (crescent); 4 h: micronuclear meiosis; 5 h: four haploid meiotic micronuclei, three of which subsequently undergo programmed nuclear death (PND); 6 h: postzygotic division; 7–8 h: macronuclear anlagen differentiation. Prior to mating, one of the parental strains was incubated with mitotracker red dye to determine the identity of each partner in mating pairs.
Figure 5.
Endoreplication and rDNA amplification during Tetrahymena development.
(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 (S1 Table). 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 BamHI and probed with an rDNA 3′ NTS probe to distinguish macronuclear B (4.0 kb) and C3 (2.5 kb) rDNA alleles.
Figure 6.
Developmental regulation of pre-RC components.
Whole cell lysates were prepared from matings between wild type strains, CU427 and CU428, at indicated time points during conjugation. 0–6 h, micronuclear DNA replication; 9–24 h, endoreplication phase I (Endo I). Mating cultures were re-fed at 24 h to complete development (endoreplication phase II; Endo II). Equivalent amounts of total protein (20 µg) were separated by denaturing polyacrylamide gel electrophoresis and subjected to western blot analysis. (B) Flow cytometry analysis samples analyzed in panel A. Nuclei were isolated and stained with propidium iodide. Each histogram represents the number of counted nuclei (x-axis) versus DNA content (y-axis). OM, old parental macronucleus, which is degraded in conjugants. (C) Western blot analysis of wild type cells (CU428) synchronized at the G1/S border by starvation for 18 h or by centrifugal elutriation of a log phase vegetative culture.
Figure 7.
Two-dimensional gel analysis of rDNA replication intermediates during development.
(A) Schematic of the palindromic rDNA 5′NTS fragment generated by HindIII (H3) digestion, and possible replication intermediate patterns resolved by neutral-neutral 2D gel electrophoresis. Pro, promoter; D1 and D2, imperfect 430 bp tandem duplications and harbor the rDNA origins of replication. Simple Y (arrow), passive replication of 5′ NTS origins. Bubble (filled arrowhead) or bubble to Y, bidirectional replication within the 5′ NTS. Composite (simple Y arc and bubbles), active and passive replication of the 5′ NTS. Double Y, two converging forks initiating within or outside the 5′ NTS. Barrier, replication of the 5′ NTS by converging forks, in which the first fork entered and terminated at a barrier prior to entry of the second fork. Aberrant Y (unfilled arrowhead), simple Y arc containing partially replicated DNA. Diagonal dashed line, migration of linear duplex DNA fragments; dotted arc, reference pattern for simple Y arc intermediates. (B) C3 rDNA amplification replication intermediates after mating for 14 and 16 h, and subsequent refeeding (at 24 h) for an additional 8 h. Mating: strains SB4202 and SB1934. (C) Mung bean nuclease (MBN) digestion on C3 rDNA amplification intermediates in matings between SB4202 and SB1934 collected after refeeding with media for 8 h. DNA samples from log phase SB4204 stain were collected as the control.