scholarly journals Incorporation of nucleotide tracers into nucleic acids in permeabilized cells and cellular homogenates

1986 ◽  
Vol 238 (1) ◽  
pp. 13-21 ◽  
Author(s):  
K Pawlak ◽  
C Lawi-Berger ◽  
W Sadée
Keyword(s):  
Dna Replication ◽  
Neuroblastoma Cells ◽  
Leukaemia Virus ◽  
Permeabilized Cells ◽  
Dna And Rna ◽  
Mouse Macrophages ◽  
Dna And Rna Synthesis ◽  
Transcription In Vitro ◽  
Cellular Dna

The validity of permeabilized cells as a model of DNA and RNA synthesis was studied with the use of mouse S-49 lymphoblastoma cells rendered permeable by exposure to L-alpha-lysophosphatidylcholine. The permeabilized cells readily incorporated exogenously supplied cytosine and uracil nucleotides into HClO4-insoluble macromolecular material. However, the incorporation of these tracers did not require the three other complementary nucleotides, and adenine, guanine or thymine nucleotide tracers were incorporated at much lower rates. These results, which were also obtained with permeabilized Abelsohn-leukaemia-virus-transformed mouse macrophages, mouse neuroblastoma cells and S-49 lymphoblastoma homogenates, are inconsistent with semi-conservative DNA replication or RNA transcription; rather, they suggest the involvement of terminal nucleotidyltransferase(s) that mediate the incorporation of uracil and cytosine nucleotides. DNA synthesis was restored when permeabilized cells or cellular homogenates were supplemented with denatured salmon testes DNA. These results suggest that endogenous cellular DNA is impaired in its function as a template for DNA replication and transcription in vitro. Metabolic channelling or compartmentation of nucleic-acid-precursor pathways could not be demonstrated in the permeabilized cells.

scholarly journals Mechanism of action of purpuromycin

1992 ◽  
Vol 284 (1) ◽  
pp. 47-52 ◽  
Author(s):  
P Landini ◽  
E Corti ◽  
B P Goldstein ◽  
M Denaro
Keyword(s):  
Protein Synthesis ◽  
Rna Synthesis ◽  
Intact Cell ◽  
Acid Synthesis ◽  
Free System ◽  
Dna And Rna ◽  
Dna And Rna Synthesis ◽  
Dependent Protein ◽  
Elongation Step

Purpuromycin, an antibiotic active against both fungi and bacteria, shows different modes of action against these two kinds of micro-organisms; in Candida albicans it inhibits RNA synthesis, whereas in Bacillus subtilis protein synthesis is primarily affected, with DNA and RNA synthesis blocked at higher concentrations of the drug. In bacterial cell-free protein-synthesis systems, purpuromycin did not inhibit synthesis from endogenous mRNA (elongation of peptides initiated within the intact cell) but inhibited MS2-phase RNA-dependent protein synthesis (which requires initiation) by 50% at 0.1 mg/l. Poly(U)-directed polyphenylalanine synthesis was 50% inhibited by 20 mg of purpuromycin/l when added to a complete system; however, when purpuromycin was preincubated with ribosomes dissociated into 30 S and 50 S subunits, the concentration for 50% inhibition fell to 0.1 mg/l. By contrast, in a C. albicans cell-free system poly(U)-directed polyphenylalanine synthesis was partially inhibited only at 200 mg/l. Purpuromycin also inhibited polynucleotide synthesis in vitro in reactions using Escherichia coli or wheat-germ RNA polymerases or E. coli DNA polymerase I. We suggest that in bacteria the primary target of purpuromycin is on ribosomes and that its action precedes the elongation step of protein synthesis. The effect on nucleic acid synthesis in both fungi and bacteria may be due to interaction of purpuromycin with DNA.

Interaction between natural compounds and human topoisomerase I

2012 ◽  
Vol 393 (11) ◽  
pp. 1327-1340 ◽  
Author(s):  
Silvia Castelli ◽  
Andrea Coletta ◽  
Ilda D’Annessa ◽  
Paola Fiorani ◽  
Cinzia Tesauro ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Dna Replication ◽  
Topoisomerase I ◽  
Rna Synthesis ◽  
Natural Compounds ◽  
Dna And Rna ◽  
Cellular Target ◽  
Dna And Rna Synthesis ◽  
Medical Interest ◽  
Different Levels

Abstract Eukaryotic topoisomerase I (Top1) is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA during important processes including DNA replication, transcription, recombination and chromosome condensation. Human Top1 I is of significant medical interest since it is the unique cellular target of camptothecin (CPT), a plant alkaloid that rapidly blocks both DNA and RNA synthesis. In this review, together with CPT, we point out the interaction between human Top1 and some natural compounds, such us terpenoids, flavonoids, stilbenes and fatty acids. The drugs can interact with the enzyme at different levels perturbing the binding, cleavage, rotation or religation processes. Here we focus on different assays that can be used to identify the catalytic step of the enzyme inhibited by different natural compounds.

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 An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication

NAR Cancer ◽  
2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Robert M Brosh ◽  
Yuliang Wu
Keyword(s):  
Dna Replication ◽  
Double Helix ◽  
Dna Helicase ◽  
Nucleic Acid Binding ◽  
Dna Helicases ◽  
Recent Advances ◽  
G4 Dna ◽  
Cellular Dna

Abstract A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.

scholarly journals A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B

2006 ◽  
Vol 175 (3) ◽  
pp. 389-400 ◽  
Author(s):  
Hiroshi Kimura ◽  
Nanako Takizawa ◽  
Eric Allemand ◽  
Tetsuya Hori ◽  
Francisco J. Iborra ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Fluorescent Protein ◽  
Histone Variants ◽  
Permeabilized Cells ◽  
Cellular Factors ◽  
Core Histones ◽  
Green Fluorescent ◽  
Histone Exchange

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.

Mammalian DNA polymerase α : a replication-competent holoenzyme form from calf thymus

1987 ◽  
Vol 317 (1187) ◽  
pp. 421-428 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Consensus Sequence ◽  
Porcine Circovirus ◽  
Chromosomal Replication ◽  
Dna Polymerase Α ◽  
Calf Thymus ◽  
Dna Replication Origins ◽  
Cellular Dna

Calf thymus DNA polymerase α, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli , can be isolated as a distinct complex. A specific multiprotein form of the polymerase α, a form designated replication-com petent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme α selectively initiates pcv DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.

DNA and RNA Synthesis by Postpubertal Undescended Testis In Vitro

1989 ◽  
Vol 22 (1) ◽  
pp. 91-94 ◽  
Author(s):  
M. Nakamura ◽  
N. Nonomura ◽  
M. Namiki ◽  
A. Okuyama ◽  
E. Koh ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Undescended Testis ◽  
Dna And Rna ◽  
Dna And Rna Synthesis
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