Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification

ABSTRACT The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

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Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

Journal of Cell Science ◽

10.1242/jcs.01405 ◽

2004 ◽

Vol 117 (22) ◽

pp. 5221-5231 ◽

Cited By ~ 38

Author(s):

M. R. Lidonnici

Keyword(s):

Cell Cycle ◽

Origin Recognition Complex ◽

Human Origin ◽

Origin Recognition

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Dynamic Localization of Protein Phosphatase Type 1 in the Mitotic Cell Cycle of Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.149.1.125 ◽

2000 ◽

Vol 149 (1) ◽

pp. 125-140 ◽

Cited By ~ 46

Author(s):

Andrew Bloecher ◽

Kelly Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Protein Phosphatase ◽

Essential Gene ◽

Mitotic Cell ◽

Type I ◽

Mitotic Cell Cycle ◽

Dependent Manner ◽

Bud Neck ◽

Protein Phosphatase Type

Protein phosphatase type I (PP1), encoded by the single essential gene GLC7 in Saccharomyces cerevisiae, functions in diverse cellular processes. To identify in vivo subcellular location(s) where these processes take place, we used a functional green fluorescent protein (GFP)–Glc7p fusion protein. Time-lapse fluorescence microscopy revealed GFP–Glc7p localizes predominantly in the nucleus throughout the mitotic cell cycle, with the highest concentrations in the nucleolus. GFP–Glc7p was also observed in a ring at the bud neck, which was dependent upon functional septins. Supporting a role for Glc7p in bud site selection, a glc7-129 mutant displayed a random budding pattern. In α-factor treated cells, GFP–Glc7p was located at the base of mating projections, again in a septin-dependent manner. At the start of anaphase, GFP–Glc7p accumulated at the spindle pole bodies and remained there until cytokinesis. After anaphase, GFP–Glc7p became concentrated in a ring that colocalized with the actomyosin ring. A GFP–Glc7-129 fusion was defective in localizing to the bud neck and SPBs. Together, these results identify sites of Glc7p function and suggest Glc7p activity is regulated through dynamic changes in its location.

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Control of DNA Rereplication via Cdc2 Phosphorylation Sites in the Origin Recognition Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5767-5777.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5767-5777 ◽

Cited By ~ 54

Author(s):

Amit Vas ◽

Winnie Mok ◽

Janet Leatherwood

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Origin Recognition Complex ◽

Safety Net ◽

Replication Initiation ◽

Initiation Factor ◽

Phosphorylation Sites ◽

Cdc2 Kinase ◽

Origin Recognition ◽

Dna Rereplication

ABSTRACT Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G2 and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

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Carvedilol Inhibits Angiotensin II-Induced Proliferation and Contraction in Hepatic Stellate Cells through the RhoA/Rho-Kinase Pathway

BioMed Research International ◽

10.1155/2019/7932046 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Ying Wu ◽

Zhen Li ◽

Sining Wang ◽

Aiyuan Xiu ◽

Chunqing Zhang

Keyword(s):

Cell Cycle ◽

Angiotensin Ii ◽

Liver Fibrosis ◽

Cell Cycle Progression ◽

Rho Kinase ◽

Cell Counting ◽

Type I ◽

Dependent Manner ◽

Ang Ii ◽

Cycle Progression

Aim. Carvedilol is a nonselective beta-blocker used to reduce portal hypertension. This study investigated the effects and potential mechanisms of carvedilol in angiotensin II- (Ang II-) induced hepatic stellate cell (HSC) proliferation and contraction. Methods. The effect of carvedilol on HSC proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell cycle progression and apoptosis in HSCs were determined by flow cytometry. A collagen gel assay was used to confirm HSC contraction. The extent of liver fibrosis in mice was evaluated by hematoxylin-eosin (H&E) and Sirius Red staining. Western blot analyses were performed to detect the expression of collagen I, collagen III, α-smooth muscle actin (α-SMA), Ang II type I receptor (AT1R), RhoA, Rho-kinase 2 (ROCK2), and others. Results. The results showed that carvedilol inhibited HSC proliferation and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Carvedilol also modulated Bcl-2 family proteins and increased apoptosis in Ang II-treated HSCs. Furthermore, carvedilol inhibited HSC contraction induced by Ang II, an effect that was associated with AT1R-mediated RhoA/ROCK2 pathway interference. In addition, carvedilol reduced α-SMA expression and collagen deposition and attenuated liver fibrosis in carbon tetrachloride (CCl4)-treated mice. The in vivo data further confirmed that carvedilol inhibited the expression of angiotensin-converting enzyme (ACE), AT1R, RhoA, and ROCK2. Conclusions. The results indicated that carvedilol dose-dependently inhibited Ang II-induced HSC proliferation by impeding cell cycle progression, thus alleviating hepatic fibrosis. Furthermore, carvedilol could inhibit Ang II-induced HSC contraction by interfering with the AT1R-mediated RhoA/ROCK2 pathway.

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DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Molecular Biology of the Cell ◽

10.1091/mbc.12.6.1671 ◽

2001 ◽

Vol 12 (6) ◽

pp. 1671-1685 ◽

Cited By ~ 83

Author(s):

Mohammed Momin Shareef ◽

Chadwick King ◽

Mona Damaj ◽

RamaKrishna Badagu ◽

Da Wei Huang ◽

...

Keyword(s):

Dna Binding ◽

Origin Recognition Complex ◽

Polytene Chromosomes ◽

Binding Activity ◽

Hmg Proteins ◽

Mitotic Chromosomes ◽

Origin Firing ◽

Dna Binding Activity ◽

Origin Recognition

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

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Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

Journal of Biological Chemistry ◽

10.1074/jbc.m111.338467 ◽

2012 ◽

Vol 287 (15) ◽

pp. 11891-11898 ◽

Cited By ~ 27

Author(s):

Kyung Yong Lee ◽

Sung Woong Bang ◽

Sang Wook Yoon ◽

Seung-Hoon Lee ◽

Jong-Bok Yoon ◽

...

Keyword(s):

Cell Cycle ◽

Origin Recognition Complex ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Replication Origins ◽

Eukaryotic Chromosome ◽

M Phase ◽

Cycle Dependent ◽

Prereplicative Complex ◽

Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

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DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence

10.1101/2020.09.27.314948 ◽

2020 ◽

Author(s):

Amanda L. Fry ◽

Amy Webster ◽

Rojin Chitrakar ◽

L. Ryan Baugh ◽

E. Jane Albert Hubbard

Keyword(s):

Cell Cycle ◽

Germ Line ◽

Primordial Germ Cells ◽

Primordial Germ Cell ◽

Gene Activation ◽

Dependent Manner ◽

Germline Gene ◽

C Elegans ◽

Zygotic Gene ◽

Zygotic Gene Activation

AbstractQuiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. In C. elegans mutant for daf-18, the sole C. elegans PTEN ortholog, primordial germ cells (PGCs) divide inappropriately in starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in zygotic germline gene activation, though not in germline fate specification. However, TOR is less important to zygotic germline gene expression, suggesting that in the absence of food daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.

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Brefeldin A-dependent Membrane Tubule Formation Reconstituted In Vitro Is Driven by a Cell Cycle–regulated Microtubule Motor

Molecular Biology of the Cell ◽

10.1091/mbc.11.3.941 ◽

2000 ◽

Vol 11 (3) ◽

pp. 941-955 ◽

Cited By ~ 18

Author(s):

Alasdair M. Robertson ◽

Victoria J. Allan

Keyword(s):

Cell Cycle ◽

Cultured Cells ◽

Brefeldin A ◽

Binding Activity ◽

Membrane Dynamics ◽

Dependent Manner ◽

Tubule Formation ◽

Early Endosomes

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus,trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5′-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of ∼100 and ∼130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

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M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6694 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6694-6701 ◽

Cited By ~ 44

Author(s):

C Caelles ◽

H Hennemann ◽

M Karin

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Protein Kinase ◽

Dna Binding ◽

Binding Activity ◽

Dependent Manner ◽

Mitotic Kinase ◽

Dna Binding Activity ◽

M Phase ◽

A Cell

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

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