scholarly journals The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7532
Author(s):  
Clive R. Bagshaw ◽  
Jendrik Hentschel ◽  
Michael D. Stone
Keyword(s):  
Time Course ◽  
Systematic Errors ◽  
Reverse Transcriptases ◽  
Rigorous Approach ◽  
Translocation Event ◽  
Nucleotide Incorporation ◽  
Standard Procedures ◽  
Nucleotide Addition ◽  
Basic Reaction ◽  
Linear Chromosomes

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

scholarly journals Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114

2005 ◽  
Vol 387 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Clara E. CASES-GONZÁLEZ ◽  
Luis MENÉNDEZ-ARIAS
Keyword(s):  
Catalytic Efficiency ◽  
Fold Increase ◽  
Polymerase Activity ◽  
Site Directed Mutagenesis ◽  
Van Der Waals Interactions ◽  
Reverse Transcriptases ◽  
Nucleotide Incorporation ◽  
Steady State Kinetic ◽  
Nucleotide Specificity ◽  
Hiv 1

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

scholarly journals Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e85270 ◽  
Author(s):  
Pawel Zajac ◽  
Saiful Islam ◽  
Hannah Hochgerner ◽  
Peter Lönnerberg ◽  
Sten Linnarsson
Keyword(s):  
Reverse Transcriptases ◽  
Nucleotide Incorporation

scholarly journals Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

2019 ◽  
Author(s):  
Alfred M. Lentzsch ◽  
Jun Yao ◽  
Rick Russell ◽  
Alan M. Lambowitz
Keyword(s):  
Nucleic Acids ◽  
Group Ii Intron ◽  
Template Switching ◽  
Reverse Transcriptases ◽  
Donor And Acceptor ◽  
Nucleotide Addition ◽  
Single Base Pair ◽  
Group Ii ◽  
Template Switch ◽  
Template Dna

ABSTRACTThe reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5’ end of one template to the 3’ end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3’-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3’-DNA overhang complementary to the 3’ nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3’-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3’ end of the acceptor template. The reliance on a single base pair with the 3’ nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3’ end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

scholarly journals Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA

2020 ◽  
Vol 48 (18) ◽  
pp. 10087-10100
Author(s):  
Song Mao ◽  
Bartosz Sekula ◽  
Milosz Ruszkowski ◽  
Srivathsan V Ranganathan ◽  
Phensinee Haruehanroengra ◽  
...  
Keyword(s):  
Base Pairing ◽  
Reverse Transcriptases ◽  
X Ray Crystallography ◽  
Coding Efficiency ◽  
Gene Replication ◽  
Nucleotide Incorporation ◽  
Duplex Stability ◽  
Rna Duplexes ◽  
Fine Tune ◽  
Hiv 1

Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

Statistics and parallaxes

1978 ◽  
Vol 48 ◽  
pp. 7-29
Author(s):  
T. E. Lutz
Keyword(s):  
Experimental Design ◽  
Statistical Methods ◽  
Review Paper ◽  
Systematic Errors ◽  
Past Experience ◽  
Random Errors ◽  
Trigonometric Parallaxes ◽  
Systematic And Random Errors

This review paper deals with the use of statistical methods to evaluate systematic and random errors associated with trigonometric parallaxes. First, systematic errors which arise when using trigonometric parallaxes to calibrate luminosity systems are discussed. Next, determination of the external errors of parallax measurement are reviewed. Observatory corrections are discussed. Schilt’s point, that as the causes of these systematic differences between observatories are not known the computed corrections can not be applied appropriately, is emphasized. However, modern parallax work is sufficiently accurate that it is necessary to determine observatory corrections if full use is to be made of the potential precision of the data. To this end, it is suggested that a prior experimental design is required. Past experience has shown that accidental overlap of observing programs will not suffice to determine observatory corrections which are meaningful.

Dielectronic Recombination in the Average-Atom Model

1988 ◽  
Vol 102 ◽  
pp. 215
Author(s):  
R.M. More ◽  
G.B. Zimmerman ◽  
Z. Zinamon
Keyword(s):  
Detailed Balance ◽  
Systematic Errors ◽  
Dielectronic Recombination ◽  
Rate Equations ◽  
Strong Correlations ◽  
Dense Plasmas ◽  
Atom Model ◽  
New Feature ◽  
Average Atom Model ◽  
Attachment Process

Autoionization and dielectronic attachment are usually omitted from rate equations for the non–LTE average–atom model, causing systematic errors in predicted ionization states and electronic populations for atoms in hot dense plasmas produced by laser irradiation of solid targets. We formulate a method by which dielectronic recombination can be included in average–atom calculations without conflict with the principle of detailed balance. The essential new feature in this extended average atom model is a treatment of strong correlations of electron populations induced by the dielectronic attachment process.

Ultrastructural Changes of the Symbiotic Chlorella-Like Alga Isolated from Hydra Viridis

1982 ◽  
Vol 40 ◽  
pp. 320-321
Author(s):  
K.W. Lee ◽  
R.H. Meints ◽  
D. Kuczmarski ◽  
J.L. Van Etten
Keyword(s):  
Time Course ◽  
Reciprocal Cross ◽  
Ultrastructural Level ◽  
Ultrastructural Changes ◽  
Symbiotic Relationship ◽  
Cell Recognition ◽  
Green Hydra ◽  
Different Strains ◽  
Hydra Viridis

The physiological, biochemical, and ultrastructural aspects of the symbiotic relationship between the Chlorella-like algae and the hydra have been intensively investigated. Reciprocal cross-transfer of the Chlorellalike algae between different strains of green hydra provide a system for the study of cell recognition. However, our attempts to culture the algae free of the host hydra of the Florida strain, Hydra viridis, have been consistently unsuccessful. We were, therefore, prompted to examine the isolated algae at the ultrastructural level on a time course.

Destruction of Actin Filaments by Osmium Tetroxide

1977 ◽  
Vol 35 ◽  
pp. 466-467
Author(s):  
P. Maupin-Szamier ◽  
T. D. Pollard
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Osmium Tetroxide ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Sodium Phosphate Buffer ◽  
Transmission Electron ◽  
The Difference ◽  
Rates Of Decay ◽  
Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

Effect of Niacin on Mitochondria of Allium Cepa Root Cells

1967 ◽  
Vol 25 ◽  
pp. 78-79
Author(s):  
M. Arif Hayat
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Hydrogen Transfer ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Nicotinamide Adenine ◽  
Root Tips ◽  
Root Cells ◽  
Transfer Processes ◽  
Standard Procedures ◽  
A Cell ◽  
Critical Interest

Although it is recognized that niacin (pyridine-3-carboxylic acid), incorporated as the amide in nicotinamide adenine dinucleotide (NAD) or in nicotinamide adenine dinucleotide phosphate (NADP), is a cofactor in hydrogen transfer in numerous enzyme reactions in all organisms studied, virtually no information is available on the effect of this vitamin on a cell at the submicroscopic level. Since mitochondria act as sites for many hydrogen transfer processes, the possible response of mitochondria to niacin treatment is, therefore, of critical interest.Onion bulbs were placed on vials filled with double distilled water in the dark at 25°C. After two days the bulbs and newly developed root system were transferred to vials containing 0.1% niacin. Root tips were collected at ¼, ½, 1, 2, 4, and 8 hr. intervals after treatment. The tissues were fixed in glutaraldehyde-OsO4 as well as in 2% KMnO4 according to standard procedures. In both cases, the tissues were dehydrated in an acetone series and embedded in Reynolds' lead citrate for 3-10 minutes.

Microstructure of Electro-Eroded Cemented Carbide Surfaces

1968 ◽  
Vol 26 ◽  
pp. 284-285
Author(s):  
V. N. Filimonenko ◽  
M. H. Richman ◽  
J. Gurland
Keyword(s):  
Surface Layer ◽  
Cobalt Content ◽  
Cemented Carbides ◽  
Face Centered Cubic ◽  
X Ray Diffraction ◽  
Metallographic Examination ◽  
Standard Procedures ◽  
Face Centered ◽  
Diamond Paste ◽  
Plastic Carbon

The high temperatures and pressures that are found in a spark gap during electrical discharging lead to a sharp phase transition and structural transformation in the surface layer of cemented carbides containing WC and cobalt. By means of X-ray diffraction both W2C and a high-temperature monocarbide of tungsten (face-centered cubic) were detected after electro-erosion. The W2C forms as a result of the peritectic reaction, WC → W2C+C. The existence and amount of the phases depend on both the energy of the electro-spark discharge and the cobalt content. In the case of a low-energy discharge (i.e. C=0.01μF, V = 300v), WC(f.c.c.) is generally formed in the surface layer. However, at high energies, (e.g. C=30μF, V = 300v), W2C is formed at the surface in preference to the monocarbide. The phase transformations in the surface layer are retarded by the presence of larger percentages of cobalt.Metallographic examination of the electro-eroded surfaces of cemented carbides was carried out on samples with 5-30% cobalt content. The specimens were first metallographically polished using diamond paste and standard procedures and then subjected to various electrical discharges on a Servomet spark machining device. The samples were then repolished and etched in a 3% NH4OH electrolyte at -0.5 amp/cm2. Two stage plastic-carbon replicas were then made and shadowed with chromium at 27°.

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