31 research outputs found
Distinct DNA Replication Programs in Tetrahymena Thermophila
DNA replication is a vital process to duplicate genetic material for inheritance. A conventional mitotic cell cycle is composed of G1, S, G2 and M phases, and DNA is replicated during S phase. Besides the conventional cell cycle, there are other distinct replication programs. Here in my dissertation, I used the organism Tetrahymena thermophila to study DNA replication because it employs alternative DNA replication programs, such as genome-wide endoreplication, locus-specific gene amplification and an unprecedented DNA replication program in cells that are recovered from hydroxyurea (HU) induced replication stress.
In my dissertation research, I determined that ribosomal DNA (rDNA) minichromosome amplification occurs when non-rDNA chromosomes are undergoing endoreplication during Tetrahymena development, and that both programs are shut down simultaneously. I found that rDNA amplification is then switched to endoreplication upon refeeding when the levels of the initiation proteins, the origin recognition complex (ORC) and the minichromosome maintenance protein complex (MCM2-7) are dramatically reduced. During this stage, the rDNA origin is not utilized, and a higher origin density was observed on a genome-wide scale. These data indicate that origin utilization is altered for endoreplication and suggest an ORC-independent initiation mechanism. More importantly, rDNA replication intermediates that are accumulated in both wild type endoreplication and vegetative S phase of a histone monomethyltransferase defective strain TXR1Δ share the same signature, suggesting epigenetic modifications may be involved in replication initiation and elongation during endoreplication.
As part of my dissertation research, I studied a DNA replication program that occurs after ORC and MCM proteins are degraded in hydroxyurea treated cells. I found that replication forks are arrested rather than slowed down upon HU treatment, when the protein levels of ORC and MCMs are degraded. I detected new origin firing on a genome-wide scale upon HU removal, before the protein levels of ORC and MCMs are restored. Moreover, the rDNA origin that is used for vegetative S phase is not utilized in this specialized replication program. The collective data suggest that an ORC-independent initiation is utilized.
In summary, my research has led to new discoveries of distinct DNA replication initiation and elongation mechanisms in eukaryotes
Developmentally Programmed Switches in DNA Replication: Gene Amplification and Genome-Wide Endoreplication in <i>Tetrahymena</i>
Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in the developing macronucleus of Tetrahymena thermophila. Subsequently, all macronuclear chromosomes replicate once per cell cycle during vegetative growth. Here, we describe an unanticipated, programmed switch in the regulation of replication initiation in the rDNA minichromosome. Early in development, the 21 kb rDNA minichromosome is preferentially amplified from 2 C to ~800 C from well-defined origins, concurrent with genome-wide endoreplication (2 C to 8–16 C) in starved mating Tetrahymena (endoreplication (ER) Phase 1). Upon refeeding, rDNA and non-rDNA chromosomes achieve their final copy number through resumption of just the endoreplication program (ER Phase 2). Unconventional rDNA replication intermediates are generated primarily during ER phase 2, consistent with delocalized replication initiation and possible formation of persistent RNA-DNA hybrids. Origin usage and replication fork elongation are affected in non-rDNA chromosomes as well. Despite the developmentally programmed 10-fold reduction in the ubiquitous eukaryotic initiator, the Origin Recognition Complex (ORC), active initiation sites are more closely spaced in ER phases 1 and 2 compared to vegetative growing cells. We propose that initiation site selection is relaxed in endoreplicating macronuclear chromosomes and may be less dependent on ORC
Developmental regulation of the Tetrahymena thermophila origin recognition complex.
The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development
Sumoylation of the DNA polymerase ε by the Smc5/6 complex contributes to DNA replication.
DNA polymerase epsilon (Pol ε) is critical for genome duplication, but little is known about how post-translational modification regulates its function. Here we report that the Pol ε catalytic subunit Pol2 in yeast is sumoylated at a single lysine within a catalytic domain insertion uniquely possessed by Pol2 family members. We found that Pol2 sumoylation occurs specifically in S phase and is increased under conditions of replication fork blockade. Analyses of the genetic requirements of this modification indicate that Pol2 sumoylation is associated with replication fork progression and dependent on the Smc5/6 SUMO ligase known to promote DNA synthesis. Consistently, the pol2 sumoylation mutant phenotype suggests impaired replication progression and increased levels of gross chromosomal rearrangements. Our findings thus indicate a direct role for SUMO in Pol2-mediated DNA synthesis and a molecular basis for Smc5/6-mediated regulation of genome stability
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 × 10<sup>5</sup> 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: 2.5×10<sup>5</sup> cells/ml).</p
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
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
Checkpoint Activation of an Unconventional DNA Replication Program in <i>Tetrahymena</i>
<div><p>The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in <i>Tetrahymena thermophila</i> in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.</p></div
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