scholarly journals Tandem DNA Repeats: Generation and Propagation in the Microgene Polymerization Reaction and in Vivo.

10.5772/13514 ◽  
2011 ◽  
Author(s):  
Mark Itsko ◽  
Eitan Ben-Dov ◽  
Avinoam Rabinovitch ◽  
Arieh Zaritsky
Keyword(s):  
Polymerization Reaction ◽  
Dna Repeats

Dynamics of Telomeric DNA Turnover in Yeast

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 63-73
Author(s):  
Michael J McEachern ◽  
Dana Hager Underwood ◽  
Elizabeth H Blackburn
Keyword(s):  
Kluyveromyces Lactis ◽  
Mutant Gene ◽  
Dna Repeats ◽  
Wild Type ◽  
Telomeric Dna ◽  
Cell Divisions ◽  
Dna Turnover ◽  
Length Regulation ◽  
Turn Over

Abstract Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

scholarly journals UvrD Helicase Suppresses Recombination and DNA Damage-Induced Deletions

2006 ◽  
Vol 188 (15) ◽  
pp. 5450-5459 ◽  
Author(s):  
Josephine Kang ◽  
Martin J. Blaser
Keyword(s):  
Dna Damage ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Critical Role ◽  
Genotoxic Stress ◽  
Recombinational Repair ◽  
Dna Repeats ◽  
H Pylori ◽  
High Level

ABSTRACT UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori, a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of inter- and intragenomic recombination and mutation. H. pylori contains numerous DNA repeats in its compact genome and inhabits an environment rich in DNA-damaging agents that can lead to increased rearrangements between such repeats. We find that H. pylori UvrD functions to repair DNA damage and limit homologous recombination and DNA damage-induced genomic rearrangements between DNA repeats. Our results suggest that UvrD and other NER pathway proteins play a prominent role in maintaining genome integrity, especially after DNA damage; thus, NER may be especially critical in organisms such as H. pylori that face high-level genotoxic stress in vivo.

scholarly journals Characterization of Flowering Genes of Arabidopsis thaliana for Mirror Repeats

2021 ◽  
Vol 12 (3) ◽  
pp. 2852-2861
Keyword(s):  
Arabidopsis Thaliana ◽  
Genetic Diseases ◽  
Dna Repeats ◽  
Quick Method ◽  
Flowering Genes ◽  
Repeat Sequences ◽  
Mutational Hotspots ◽  
And Function

A variety of simple DNA repeats are enriched in the eukaryotic genomes. Recent studies have proven their importance in understanding genome organization and function, especially how genomes evolve using them as mutational hotspots during DNA replication. Mirror repeat sequences, the most underrated subset of this class of repeats, are now gaining importance because of their probable involvement in developing several genetic diseases in humans. These repeats typically adopt H-DNA conformations in both in-vitro and in-vivo conditions. On the other end, plants were still not analyzed for their presence or distribution and whether they are responsible for causing diseases in them or not. The present study aims to extract mirror repeats in the flowering genes of Arabidopsis thaliana. To this end, we have deployed FPCB (FASTA-PARALLEL COMPLEMENT-BLAST), an efficacious and quick method to extract perfect and degenerate mirror repeat sequences through pattern matching of alignments with user-defined algorithmic parameters. All the analyzed genes were reported to have quite high densities of mirror sequences. A total of 93 unique mirror repeats of significant lengths were extracted in the analyzed genes.

scholarly journals Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Astrid Lancrey ◽  
Alexandra Joubert ◽  
Evelyne Duvernois-Berthet ◽  
Etienne Routhier ◽  
Saurabh Raj ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Nucleosome Occupancy ◽  
Nucleosome Positioning ◽  
Dna Repeats ◽  
Repeat Array ◽  
Synthetic Dna

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

Evidence for Nucleosomal Phasing and a Novel Protein Specifically Binding to Cucumber Satellite DNA

1994 ◽  
Vol 49 (1-2) ◽  
pp. 79-86 ◽  
Author(s):  
Thilo C. Fischer ◽  
Sabine Groner ◽  
Ulrike Zentgraf ◽  
Vera Hemleben
Keyword(s):  
Satellite Dna ◽  
Specific Binding ◽  
Apparent Molecular Weight ◽  
Micrococcal Nuclease ◽  
Type I ◽  
Dna Repeats ◽  
Cucumis Sativus L ◽  
Nuclease Digestion

The nucleosomal organization and the protein-binding capability of highly repeated and methylated satellite DNA of cucumber (Cucumis sativus L.), comprising approx. 30% of the genome, were analyzed. Nucleosomal core DNA from satellite type I was prepared after micrococcal nuclease digestion of chromatin and sequenced. Most of the core sequences obtained could be grouped in two main (A and B) and two minor groups (C and D) indicating a specific and complex phasing of nucleosomes on this satellite DNA. In vitro, gel retardation assays with cloned satellite DNA repeats (types I-IV) demonstrated a specific binding of nuclear proteins. These specific binding effects are also obtained with genomic, in vivo methylated and sequence heterogeneous (1 to 10% diversity) satellite type I DNA. For the first time in plants, a satellite DNA-binding protein with an apparent molecular weight of 14 kDa (SAT 14) was identified.

scholarly journals In Vivo Animal Study of a Highly Viscous N-butyl Cyanoacrylate Medical Adhesive for Intravenous Embolization

Materials ◽  
2021 ◽  
Vol 14 (13) ◽  
pp. 3527
Author(s):  
Jae-Won Seo ◽  
Habeen Park ◽  
Dogeun Kim ◽  
Seoyun Lee ◽  
Young Gook Koh ◽  
...  
Keyword(s):  
Foreign Body Reaction ◽  
Vascular Occlusion ◽  
High Viscosity ◽  
Polymerization Reaction ◽  
Embolic Agent ◽  
Low Viscosity ◽  
Vein Occlusion ◽  
Lower Heat ◽  
Medical Adhesive

N-butyl cyanoacrylate (NBCA) is a liquid monomer that undergoes an exothermic polymerization reaction to form a solid upon initiation with hydroxyl anions. Recently, EGpresto, a highly viscous NBCA-based adhesive, has been developed for vascular-occlusion purposes. In this study, we investigated the heat of polymerization of EGpresto and compared the results with those of a low-viscosity NBCA glue. Results show that EGpresto exhibited a lower heat of polymerization (64 ± 7 °C vs. 34 ± 1 °C). This was due to its high viscosity, which resulted in a delayed polymerization time. To investigate the efficacy and safety of EGpresto for intravenous embolization, a 14 d in vivo animal test was conducted using three pigs. Five cc of EGpresto was injected into the epigastric vein of each animal. Complete postoperative vein occlusion was confirmed at 7 and 14 d by ultrasonographic visualization. After the animals were sacrificed, the operated and unoperated veins were exposed, and the injected adhesive was found without migration. During the histology, the injected adhesive was not found in the inner or outer vein walls, and the immune reactions seemed to be the only foreign-body reaction, showing that EGpresto is a non-toxic and safe intravascular embolic agent.

Sequence-specific DNA primer effects on telomerase polymerization activity

1993 ◽  
Vol 13 (10) ◽  
pp. 6586-6599
Author(s):  
M S Lee ◽  
E H Blackburn
Keyword(s):  
Tetrahymena Thermophila ◽  
Kinetic Studies ◽  
Polymerization Reaction ◽  
Telomeric Dna ◽  
Base Pairs ◽  
Specific Effects ◽  
Template Sequence ◽  
Catalytic Rate ◽  
Mechanistic Basis

The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo.

scholarly journals Telomere DNA G-quadruplex folding within actively extending human telomerase

2019 ◽  
Vol 116 (19) ◽  
pp. 9350-9359 ◽  
Author(s):  
Linnea I. Jansson ◽  
Jendrik Hentschel ◽  
Joseph W. Parks ◽  
Terren R. Chang ◽  
Cheng Lu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Series Data ◽  
Resonance Energy ◽  
Series Data ◽  
Dna Repeats ◽  
Telomere Repeat ◽  
G Quadruplex ◽  
Dna Primers

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

scholarly journals E-MAP-115 (ensconsin) associates dynamically with microtubules in vivo and is not a physiological modulator of microtubule dynamics

1999 ◽  
Vol 112 (23) ◽  
pp. 4243-4255 ◽  
Author(s):  
K. Faire ◽  
C.M. Waterman-Storer ◽  
D. Gruber ◽  
D. Masson ◽  
E.D. Salmon ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Polymerization Reaction ◽  
Physiological Level ◽  
Microtubule Binding ◽  
Binding Function ◽  
Associated Proteins ◽  
Green Fluorescent

Microtubule-associated proteins (MAPs) have been hypothesized to regulate microtubule dynamics and/or functions. To test hypotheses concerning E-MAP-115 (ensconsin) function, we prepared stable cell lines expressing conjugates in which the full-length MAP (Ensc) or its microtubule-binding domain (EMTB) was conjugated to one or more green fluorescent protein (GFP) molecules. Because both distribution and microtubule-binding properties of GFP-Ensc, GFP-EMTB, and 2x, 3x, or 4xGFP-EMTB chimeras all appeared to be identical to those of endogenous E-MAP-115 (ensconsin), we used the 2xGFP-EMTB molecule as a reporter for the behavior and microtubule-binding function of endogenous MAP. Dual wavelength time-lapse fluorescence imaging of 2xGFP-EMTB in cells microinjected with labeled tubulin revealed that this GFP-MAP chimera associated with the lattice of all microtubules immediately upon polymerization and dissociated concomitant with depolymerization, suggesting that dynamics of MAP:microtubule interactions were at least as rapid as tubulin:microtubule dynamics in the polymerization reaction. Presence of both GFP-EMTB chimeras and endogenous E-MAP-115 (ensconsin) along apparently all cellular microtubules at all cell cycle stages suggested that the MAP might function in modulating stability or dynamics of microtubules, a capability shown previously in transiently transfected cells. Although cells with extremely high expression levels of GFP-EMTB chimera exhibited stabilized microtubules, cells expressing four to ten times the physiological level of endogenous MAP exhibited microtubule dynamics indistinguishable from those of untransfected cells. This result shows that E-MAP-115 (ensconsin) is unlikely to function as a microtubule stabilizer in vivo. Instead, this MAP most likely serves to modulate microtubule functions or interactions with other cytoskeletal elements.

scholarly journals An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence.

Genetics ◽  
1988 ◽  
Vol 118 (2) ◽  
pp. 181-191
Author(s):  
J G de Boer ◽  
L S Ripley
Keyword(s):  
Bacteriophage T4 ◽  
Consensus Sequence ◽  
Dna Repeats ◽  
Klenow Fragment ◽  
Purine Base ◽  
Vitro Assay ◽  
Reading Frame ◽  
Dna Primers

Abstract The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

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