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. 2009 Feb 4;28(3):223-33.
doi: 10.1038/emboj.2008.282. Epub 2009 Jan 15.

Differential Targeting of Tetrahymena ORC to Ribosomal DNA and non-rDNA Replication Origins

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Free PMC article

Differential Targeting of Tetrahymena ORC to Ribosomal DNA and non-rDNA Replication Origins

Taraka R Donti et al. EMBO J. .
Free PMC article

Abstract

The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis-acting replication determinant--the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non-rDNA fragment containing two closely associated replicators, ARS1-A (0.8 kb) and ARS1-B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA-independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non-rDNA ARS1 chromosome changed across the cell cycle. In G(2) phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1-A/B. Remarkably, ORC and Mcm6 associated with just the ARS1-A replicator in G(1) phase when pre-replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non-rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.

Figures

Figure 1
Figure 1
Organization of rDNA and non-rDNA macronuclear chromosomes. (A) Schematic of the palindromic rDNA minichromosome. Expanded view of the 5′ NTS includes positioned nucleosomes (ovals) type I elements (black rectangles), pause site elements (grey rectangles), rRNA promoter (thin arrow) and replication origins (ori) which reside in the 430 bp imperfect duplicated sequences, domains 1 and 2 (thick arrows). The sequence of the type IB element T-rich strand and flanking DNA are shown, including predicted base pair interactions with 26T RNA. (B) Chromosome reorganization and replication during macronuclear development. A portion of the micronuclear chromosome encoding the single copy rRNA gene is shown (CBS: chromosome-breakage sequences). See text for additional details.
Figure 2
Figure 2
Isolation of non-rDNA replicators. (A) Schematic of the plasmid shuttle assay used to isolate Tetrahymena ARS1. DpnI-sensitive (DpnIs) plasmids containing T. thermophila genomic DNA were co-transformed into Tetrahymena. Plasmid DNA from Tetrahymena transformants was re-introduced into E. coli. ARS-containing plasmids should be DpnI-resistant (DpnIr) following replication in Tetrahymena, and can be recovered by re-transforming E. coli. (B) Ethidium bromide negative stain image of BamHI-digested plasmid DNA from two clones obtained from the plasmid shuttle assay (Vector: plasmid backbone; Insert: Tetrahymena sequences). (C) Southern blot of undigested Tetrahymena genomic DNA from ‘en masse' transformations using intact ARS1 or a derivative containing just the internal NheI fragment (see Figure 3A for details on the NheI2 derivative). DNA was isolated from pmr co-transformants propagated for 25 generations. ARS1input plasmid DNA (+) was used as a Southern blotting control. Probe: radiolabelled pCC1FOS (no insert). (D) Southern blot of BamHI-digested DNA from wild type Tetrahymena (WT, CU428) and ‘en masse' ARS1 co-transformants propagated for up to 60 generations (gen). Probe: radiolabelled ARS1 insert.
Figure 3
Figure 3
ARS1 deletion mapping. (A) Upper schematic: predicted genes (arrows) and intergenic (IG-1, IG-2) segments in the chromosomal ARS1 interval. The end points of deleted (Δ) or retained sequences in ARS1 plasmid derivatives are indicated below. Asterisks denote fragments that were subcloned into pSMART. The remaining inserts are in the original pCC1FOS vector backbone. (B) Southern blot analysis of ARS1 transformants and five of the seven depicted deletion derivatives. DNA was prepared from ‘en masse' co-transformants at defined intervals (generations: gen). BamHI-digested samples were probed with ARS1 fragments containing just those sequences present in the plasmid under examination. C: endogenous chromosomal DNA BamHI fragment; P: plasmid-derived BamHI fragment.
Figure 4
Figure 4
Deletion mapping of the ARS1-A replicator. (A) RT–PCR analysis of the ARS1-A interval. The diagram shows the relative positions of forward (F1, F4) and reverse complementary (R2–R6) primers, intron 1, and start (ATG) and stop (TGA) codons in the predicted gene. PCRs were performed on genomic DNA (G), reverse transcribed RNA (cDNA, C) and total RNA (R). A negative image of the ethidium bromide-stained gel is shown. (B) Lower panel: RT–PCR analysis of RNA from cells synchronized by centrifugal elutriation (RT primer R6, PCR primers F4 and R5). Log: RNA from an asynchronous cell culture. Upper panel: flow cytometry profile of elutriated cells at defined culturing intervals (min). (C) Southern blot analysis of NheIL-L8 and NheIL-L6 transformants, and three of the six tested NheIL-L8 deletion derivatives (see Table I). DNA was prepared from pmr ‘en masse' ARS1 co-transformants at defined intervals (generations: gen). BamHI-digested DNA was probed with ARS1 fragments containing just those sequences present in the plasmid under examination. Arrow: plasmid-derived ARS1A fragment.
Figure 5
Figure 5
ORC targeting to rDNA and non-rDNA replicators. (A) Orc1p ChIP analysis of the rDNA domain 1 origin (O), promoter (P) and coding (C) regions. ChIP was performed on T. thermophila strain TD102 using an antibody directed against the protein A IgG-binding epitope-tag in Orc1p. I: total input DNA; (−): no antibody ChIP control; (+): Orc1p ChIP pellet. (B) Orc1p ChIP analysis of the 60 kb segment spanning ARS1 in the endogenous macronuclear chromosome (strain TD102). PCR products derived from primer sets A1 to A12 are spaced at ∼5 kb intervals. (C, D) Streptavidin (SA) chromatin pull-down analysis of rDNA and ARS1 chromosome intervals. Strain MM201 produces a 26T RNA variant that bears a sequence tag extension (Ext), whereas MM202 expresses an aptamer-tagged (Apt) 26T RNA derivative that binds to streptavidin. I: total input DNA; (−) uncoupled sepharose chromatin pull-down pellet, (+): SA-sepharose chromatin pull-down pellet. (E) ChIP analysis of the ARS1 interval with Tetrahymena Mcm6p antibodies.
Figure 6
Figure 6
Cell cycle-regulated ORC binding to the ARS1-A chromosomal locus. A log phase Tetrahymena culture was subjected to centrifugal elutriation. The G1 fraction was further cultured and samples harvested at defined intervals (min) for western blotting of Orc1p (Supplementary Figure S4), flow cytometry and ChIP. (A) Flow cytometry profiles of re-fed cultures (the left line demarcates the G1 propidium iodide (PI) peak, and the right line marks the G2 peak). (B) Orc1p cell cycle ChIP analysis (see Figure 5B for PCR primers locations). I: input; (−): no antibody ChIP pellet; (+): Orc1p ChIP pellet. (C) Orc1p ChIP analysis of the rDNA origin (O), promoter (P) and rRNA coding (C) regions (see Figure 5A schematic).
Figure 7
Figure 7
Model for ORC binding to rDNA and non-rDNA chromosomes. (A) ORC binding to the rDNA is restricted to the origin region, and occurs throughout the cell cycle due to RNA–DNA base pairing interactions between 26T RNA and rDNA origin type I element (T-rich strand). Degradation of Orc1p generates a sub-complex that remains bound to the origin. (B) ORC binding to non-rDNA replicators is independent 26T-RNA/DNA base pairing and is cell cycle regulated. In this case, the entire ORC complex dissociates from the origin upon Orc1p degradation. The holocomplex randomly binds newly synthesized daughter chromosomes and re-localizes to the preferred initiation site prior to the next S phase.

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