scholarly journals S-Phase Progression Mediates Activation of a Silenced Gene in Synthetic Nuclei

2000 ◽  
Vol 20 (11) ◽  
pp. 4169-4180 ◽  
Author(s):  
Alison J. Crowe ◽  
Julie L. Piechan ◽  
Ling Sang ◽  
Michelle C. Barton
Keyword(s):  
Dna Replication ◽  
Developmental Stage ◽  
Expression Patterns ◽  
Marker Gene ◽  
Nuclear Extract ◽  
S Phase ◽  
Developmental Expression ◽  
Aberrant Expression ◽  
Transcription Repressors

ABSTRACT Aberrant expression of developmentally silenced genes, characteristic of tumor cells and regenerating tissue, is highly correlated with increased cell proliferation. By modeling this process in vitro in synthetic nuclei, we find that DNA replication leads to deregulation of established developmental expression patterns. Chromatin assembly in the presence of adult mouse liver nuclear extract mediates developmental stage-specific silencing of the tumor marker gene alpha-fetoprotein (AFP). Replication of silenced AFP chromatin in synthetic nuclei depletes sequence-specific transcription repressors, thereby disrupting developmentally regulated repression. Hepatoma-derived factors can target partial derepression of AFP, but full transcription activation requires DNA replication. Thus, unscheduled entry into S phase directly mediates activation of a developmentally silenced gene by (i) depleting developmental stage-specific transcription repressors and (ii) facilitating binding of transactivators.

scholarly journals A Coordinated Temporal Interplay of Nucleosome Reorganization Factor, Sister Chromatin Cohesion Factor, and DNA Polymerase α Facilitates DNA Replication

2004 ◽  
Vol 24 (21) ◽  
pp. 9568-9579 ◽  
Author(s):  
Yanjiao Zhou ◽  
Teresa S.-F. Wang
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
S Phase ◽  
Terminal Region ◽  
Replication Complex ◽  
Dna Polymerase Α ◽  
Chromatid Cohesion ◽  
Phase Progression

ABSTRACT DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase α (Polα). The catalytic subunit of budding yeast Polα (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polα (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polα protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

scholarly journals Identification of DHX9 as a cell cycle regulated nucleolar recruitment factor for CIZ1

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Urvi Thacker ◽  
Tekle Pauzaite ◽  
James Tollitt ◽  
Maria Twardowska ◽  
Charlotte Harrison ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Zinc Finger Protein ◽  
S Phase ◽  
Cycle Progression ◽  
Inactive X Chromosome ◽  
Functional Characterisation ◽  
Interaction Partners

Abstract CIP1-interacting zinc finger protein 1 (CIZ1) is a nuclear matrix associated protein that facilitates a number of nuclear functions including initiation of DNA replication, epigenetic maintenance and associates with the inactive X-chromosome. Here, to gain more insight into the protein networks that underpin this diverse functionality, molecular panning and mass spectrometry are used to identify protein interaction partners of CIZ1, and CIZ1 replication domain (CIZ1-RD). STRING analysis of CIZ1 interaction partners identified 2 functional clusters: ribosomal subunits and nucleolar proteins including the DEAD box helicases, DHX9, DDX5 and DDX17. DHX9 shares common functions with CIZ1, including interaction with XIST long-non-coding RNA, epigenetic maintenance and regulation of DNA replication. Functional characterisation of the CIZ1-DHX9 complex showed that CIZ1-DHX9 interact in vitro and dynamically colocalise within the nucleolus from early to mid S-phase. CIZ1-DHX9 nucleolar colocalisation is dependent upon RNA polymerase I activity and is abolished by depletion of DHX9. In addition, depletion of DHX9 reduced cell cycle progression from G1 to S-phase in mouse fibroblasts. The data suggest that DHX9-CIZ1 are required for efficient cell cycle progression at the G1/S transition and that nucleolar recruitment is integral to their mechanism of action.

scholarly journals MAMMALIAN CHROMOSOMES IN VITRO

1964 ◽  
Vol 23 (1) ◽  
pp. 53-62 ◽  
Author(s):  
T. C. Hsu
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Sex Chromosomes ◽  
Chinese Hamster ◽  
Distal Portion ◽  
S Phase ◽  
Chromosome 1 ◽  
Chromosome 6 ◽  
Chromosome 2

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H3-thymidine labeling and the method of 5-fluorodeoxyuridine block with H3-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication

1999 ◽  
Vol 73 (4) ◽  
pp. 3004-3013 ◽  
Author(s):  
Inga Reynisdóttir ◽  
Subarna Bhattacharyya ◽  
Dong Zhang ◽  
Carol Prives
Keyword(s):  
Dna Replication ◽  
Potential Effect ◽  
T Antigen ◽  
S Phase ◽  
Large T Antigen ◽  
Murine Cell ◽  
Cell Extracts ◽  
Cdk Phosphorylation ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G1 to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (dl141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals Characterization of Mice Deficient in the Src Family Nonreceptor Tyrosine Kinase Frk/rak

2002 ◽  
Vol 22 (14) ◽  
pp. 5235-5247 ◽  
Author(s):  
Subhashini Chandrasekharan ◽  
Ting Hu Qiu ◽  
Nawal Alkharouf ◽  
Kelly Brantley ◽  
James B. Mitchell ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Expression Patterns ◽  
Developmental Expression ◽  
Epithelial Tissue ◽  
Intestinal Damage ◽  
Specific Expression ◽  
Nonreceptor Tyrosine Kinase ◽  
Euthyroid Sick Syndrome ◽  
Epithelial Tissues

ABSTRACT Frk/rak belongs to a novel family of Src kinases with epithelial tissue-specific expression. Although developmental expression patterns and functional overexpression in vitro have associated these kinases with growth suppression and differentiation, their physiological functions remain largely unknown. We therefore generated mice carrying a null mutation in iyk, the mouse homolog of Frk/rak. We report here that frk/rak−/− mice are viable, show similar growth rates to wild-type animals, and are fertile. Furthermore, a 2-year study of health and survival did not identify differences in the incidence and spectrum of spontaneous tumors or provide evidence of hyperplasias in frk/rak−/− epithelial tissues. Histological analysis of organs failed to reveal any morphological changes in epithelial tissues that normally express high levels of Frk/rak. Ultrastructural analysis of intestinal enterocytes did not identify defects in brush border morphology or structural polarization, demonstrating that Frk/rak is dispensable for intestinal cytodifferentiation. Additionally, frk/rak-null mice do not display altered sensitivity to intestinal damage induced by ionizing radiation. cDNA microarray analysis revealed an increase in c-src expression and identified subtle changes in the expression of genes regulated by thyroid hormones. Significant decreases in the circulating levels of T3 but not T4 hormone are consistent with this observation and reminiscent of euthyroid sick syndrome, a stress-associated clinical condition.

scholarly journals Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

2011 ◽  
Vol 193 (6) ◽  
pp. 995-1007 ◽  
Author(s):  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Deoxyribonucleic Acid ◽  
S Phase ◽  
Human Cells ◽  
Target Sequence ◽  
Cyclin Dependent Kinase ◽  
Functional Importance ◽  
Interacting Protein ◽  
Egg Extracts

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

scholarly journals The Schizosaccharomyces pombe cdt2+ Gene, a Target of G1-S Phase-Specific Transcription Factor Complex DSC1, Is Required for Mitotic and Premeiotic DNA Replication

Genetics ◽  
2003 ◽  
Vol 164 (3) ◽  
pp. 881-893 ◽  
Author(s):  
Shu-hei Yoshida ◽  
Hiba Al-Amodi ◽  
Taro Nakamura ◽  
Christopher J McInerny ◽  
Chikashi Shimoda
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Mitotic Cell ◽  
Life Cycles ◽  
S Phase ◽  
Growth Defect ◽  
Replication Checkpoint ◽  
Synthetically Lethal

Abstract We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1+/cdt2+ was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2+ was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2+ expression in vivo, and a cdt2+ promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30° and were unable to grow at 19°. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22+, a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19°. These observations suggest that cdt2+ plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.

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