scholarly journals 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.

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 Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

1996 ◽  
Vol 16 (1) ◽  
pp. 66-75 ◽  
Author(s):  
D Gilley ◽  
E H Blackburn
Keyword(s):  
Active Site ◽  
Tetrahymena Thermophila ◽  
Telomerase Rna ◽  
Specific Interactions ◽  
Wild Type ◽  
Specific Primer ◽  
Telomeric Dna ◽  
Template Sequence ◽  
New Evidence

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

scholarly journals Disrupting progression of the yeast Hsp90 folding pathway at different transition points results in client-specific maturation defects

Genetics ◽  
2021 ◽  
Author(s):  
Kaitlyn Hohrman ◽  
Davi Gonçalves ◽  
Kevin A Morano ◽  
Jill L Johnson
Keyword(s):  
Heat Shock Protein 90 ◽  
Multiple Roles ◽  
Folding Pathway ◽  
Temperature Sensitive ◽  
Growth Defects ◽  
Specific Effects ◽  
Client Proteins ◽  
Transition Points ◽  
Mechanistic Basis

Abstract The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad aspects of cellular physiology. Amino acid alterations located throughout Saccharomyces cerevisiae Hsp90 have been shown to result in reduced client activity and temperature-sensitive growth defects. Although some Hsp90 mutants have been shown to affect activity of particular clients more than others, the mechanistic basis of client-specific effects is unknown. We found that Hsp90 mutants that disrupt the early step of Hsp70 and Sti1 interaction, or show reduced ability to adopt the ATP-bound closed conformation characterized by Sba1 and Cpr6 interaction, similarly disrupt activity of three diverse clients, Utp21, Ssl2, and v-src. In contrast, mutants that appear to alter other steps in the folding pathway had more limited effects on client activity. Protein expression profiling provided additional evidence that mutants that alter similar steps in the folding cycle cause similar in vivo consequences. Our characterization of these mutants provides new insight into how Hsp90 and cochaperones identify and interact with diverse clients, information essential for designing pharmaceutical approaches to selectively inhibit Hsp90 function.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Insulin secretion in the spiny mouse (Acomys cahirinus). Dose and time kinetic studies with glucose in vivo and in vitro

Diabetes ◽  
1975 ◽  
Vol 24 (12) ◽  
pp. 1094-1100 ◽  
Author(s):  
A. Rabinovitch ◽  
A. Gutzeit ◽  
A. E. Renold ◽  
E. Cerasi
Keyword(s):  
Insulin Secretion ◽  
Kinetic Studies ◽  
Spiny Mouse ◽  
Acomys Cahirinus

New agents for thromboprotection

2015 ◽  
Vol 35 (04) ◽  
pp. 338-350 ◽  
Author(s):  
L. Labberton ◽  
E. Kenne ◽  
T. Renné
Keyword(s):  
Current Knowledge ◽  
Thrombus Formation ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Bleeding Tendency ◽  
New Agents ◽  
Mechanistic Basis ◽  
Normal Hemostasis ◽  
Coagulation Pathway

SummaryBlood coagulation is essential for hemostasis, however excessive coagulation can lead to thrombosis. Factor XII starts the intrinsic coagulation pathway and contact-induced factor XII activation provides the mechanistic basis for the diagnostic aPTT clotting assay. Despite its function for fibrin formation in test tubes, patients and animals lacking factor XII have a completely normal hemostasis. The lack of a bleeding tendency observed in factor XII deficiency states is in sharp contrast to deficiencies of other components of the coagulation cascade and factor XII has been considered to have no function for coagulation in vivo. Recently, experimental animal models showed that factor XII is activated by an inorganic polymer, polyphosphate, which is released from procoagulant platelets and that polyphosphate-driven factor XII activation has an essential role in pathologic thrombus formation. Cumulatively, the data suggest to target polyphosphate, factor XII, or its activated form factor XIIa for anticoagulation. As the factor XII pathway specifically contributes to thrombosis but not to hemostasis, interference with this pathway provides a unique opportunity for safe anticoagulation that is not associated with excess bleeding.The review summarizes current knowledge on factor XII functions, activators and inhibitors.

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 Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

2015 ◽  
Vol 14 (8) ◽  
pp. 817-833 ◽  
Author(s):  
Santosh Kumar ◽  
Joseph S. Briguglio ◽  
Aaron P. Turkewitz
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Content Type ◽  
Sequential Processing ◽  
Cysteine Cathepsin ◽  
Endoproteolytic Cleavage ◽  
Extracellular Stimuli ◽  
Peptide Secretion ◽  
Processing Steps

ABSTRACT In many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila , efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 ( CTH3 ). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4 results in cells (Δ cth4 ) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, Δ cth4 cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the Δ cth4 defects by mutating conserved catalytic residues, indicating that the in vivo function of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The Δ cth4 defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates.

scholarly journals Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27

1999 ◽  
Vol 19 (6) ◽  
pp. 4143-4152 ◽  
Author(s):  
Julie Parenteau ◽  
Raymund J. Wellinger
Keyword(s):  
Growth Arrest ◽  
Dna Lesions ◽  
Restrictive Temperature ◽  
Telomeric Dna ◽  
Single Stranded Dna ◽  
Template Strand ◽  
Okazaki Fragments ◽  
A Cell ◽  
Lagging Strand

ABSTRACT The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the processing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of single-stranded DNA of the template strand for lagging-strand synthesis. We examined this prediction by analyzing the terminal DNA structures generated during telomere replication in rad27strains. The lengths of the telomeric repeat tracts were found to be destabilized in rad27 strains, indicating that naturally occurring direct repeats are subject to tract expansions and contractions in such strains. Furthermore, abnormally high levels of single-stranded DNA of the templating strand for lagging-strand synthesis were observed in rad27 cells. Overexpression of Dna2p in wild-type cells also yielded single-stranded DNA regions on telomeric DNA and caused a cell growth arrest phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonuclease Exo1p alleviated the growth arrest induced by both conditions, overexpression of Dna2p and incubation of rad27cells at 37°C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not prevented by the overexpression of Exo1p in these strains, suggesting that this nuclease is not simply redundant with Rad27p. Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging-strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.

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