Polymerization Of Nucleic Acids Research Articles

Step-by-Step Towards Biological Homochirality - from Prebiotic Randomness To Perfect Asymmetry.

The history of life's formation and the origin of its stereochemistry are nearly as multifaceted as the life itself. In this review, we focus on analyzing the step-by-step path leading to what we can define as "life" in parallel to what we know about the emergence of enantiomeric imbalance and subsequent transition to full homochirality. We start at the level of assembly of the building blocks of life from inorganic molecules and build up to the polymerization and formation of nucleic acids and peptides. We report and analyze different theories at various stages of this development and try to elucidate the most plausible theory.

Parallel path mechanisms lead to nonmonotonic force-velocity curves and an optimum load for molecular motor function.

Molecular motors convert chemical potential energy into mechanical work and perform a great number of critical biological functions. Examples include the polymerization and manipulation of nucleic acids, the generation of cellular motility and contractility, the formation and maintenance of cell shape, and the transport of materials within cells. The mechanisms underlying these molecular machines are varied, but are almost always considered in the context of a single kinetic pathway that describes motor stepping. However, the multidimensional nature of protein energy landscapes suggests the possibility of multiple reaction pathways connecting two states. Here we investigate the properties of a hypothetical molecular motor able to utilize parallel translocation mechanisms. We explore motor velocity and force dependence as a function of the energy landscape of each path and reveal the potential for such a mechanism to result in negative differential conductance. More specifically, regimes exist where increasing opposing force leads to increased velocity and an optimum load for motor function. We explore how the presence of this optimum depends on the rates of the individual paths and show that the distribution of stepping times characterized by the randomness parameter may be used to test for parallel path mechanisms. Last, we caution that experimental data consisting solely of measurements of velocity as a function of ATP concentration and force cannot be used to eliminate the possibility of such a parallel path mechanism.

Concurrent Prebiotic Formation of Nucleoside-Amidophosphates and Nucleoside-Triphosphates Potentiates Transition from Abiotic to Biotic Polymerization.

Polymerization of nucleic acids in biology utilizes 5'-nucleoside triphosphates (NTPs) as substrates. The prebiotic availability of NTPs has been unresolved and other derivatives of nucleoside-monophosphates (NMPs) have been studied. However, this latter approach necessitates a change in chemistries when transitioning to biology. Herein we show that diamidophosphate (DAP), in a one-pot amidophosphorylation-hydrolysis setting converts NMPs into the corresponding NTPs via 5'-nucleoside amidophosphates (NaPs). The resulting crude mixture of NTPs are accepted by proteinaceous- and ribozyme-polymerases as substrates for nucleic acid polymerization. This phosphorylation also operates at the level of oligonucleotides enabling ribozyme-mediated ligation. This one-pot protocol for simultaneous generation of NaPs and NTPs suggests that the transition from prebiotic-phosphorylation and oligomerization to an enzymatic processive-polymerization can be more continuous than previously anticipated.

Signal Extraction, Transformation, and Magnification for Ultrasensitive and Specific Detection of Nucleic Acids.

Nucleic acid noises caused by the background and nonspecificity amplifications can jeopardize accurate polymerization and detection of nucleic acids, especially when they are analyzed in low copies. We hypothesize to reduce the noises by designing a system for specific signal extraction, transformation, and magnification to improve the specificity and sensitivity. Herein, by developing an extractor-trigger complex (ET-Combo) for the system, we have established isothermal and hybridizing combined amplifications: a one-pot detection system with two-step amplification coupled by ET-Combo. To our surprise, the signal extraction is only successful when ET-Combo is included in the first amplification. Our signal extracting, filtering, and relaying system with ET-Combo is rapid and specific, removing the noises generated during the isothermal amplification under elevated temperatures. To match the first amplification, we have designed and established a hybridizing chain reaction at high temperature. This one-pot system can resist disruption of background noises and allow detection of DNA up to five copies (single digit). With the high sensitivity, specificity, and noise resistance, our system has been successfully used to diagnose clinical samples of human papillomavirus (HPV) with the genotyping specificity.

A Parallel Ratchet-Stroke Mechanism Leads to an Optimum Force for Molecular Motor Function

Molecular motors convert chemical potential energy into mechanical work and perform a great number of critical biological functions. Examples include the polymerization and manipulation of nucleic acids, the generation of cellular motility and contractility, the formation and maintenance of cell shape, and the transport of materials within cells. The mechanisms underlying these molecular machines are routinely divided into two categories: Brownian ratchet and power stroke. While a ratchet uses chemical energy to bias thermally activated motion, a stroke depends on a direct coupling between chemical events and motion.

RNA-Catalyzed Polymerization of Deoxyribose, Threose, and Arabinose Nucleic Acids.

An RNA-dependent RNA polymerase ribozyme that was highly optimized through in vitro evolution for the ability to copy a broad range of template sequences exhibits promiscuity toward other nucleic acids and nucleic acid analogues, including DNA, threose nucleic acid (TNA), and arabinose nucleic acid (ANA). By operating on various RNA templates, the ribozyme catalyzes multiple successive additions of DNA, TNA, or ANA monomers, although with reduced efficiency compared to RNA monomers. The ribozyme can also copy DNA or TNA templates to complementary RNAs, and to a lesser extent it can operate when both the template and product strands are composed of DNA, TNA, or ANA. These results suggest that polymerase ribozymes, which are thought to have replicated RNA genomes during the early history of life, could have transferred RNA-based genetic information to and from DNA, enabling the emergence of DNA genomes prior to the emergence of proteins. In addition, genetic systems based on nucleic acid-like molecules, which have been proposed as precursors or contemporaries of RNA-based life, could have been operated upon by a promiscuous polymerase ribozyme, thus enabling the evolutionary transition between early genetic systems.

Spatiotemporal control of coacervate formation within liposomes

Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (β-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.

Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

Adsorption of nucleotides onto Fe–Mg–Al rich swelling clays

Mineral surfaces may have played a role in the origin of the first biopolymers, by concentrating organic monomers from a dilute ocean. Swelling clays provide a high surface area for the concentration of prebiotic monomers, and have therefore been the subject of numerous investigations. In that context, montmorillonite, the most abundant swelling clay in modern environments, has been extensively studied with regard to adsorption and polymerization of nucleic acids. However, montmorillonite was probably rather marginal on the primitive ocean floor compared to iron–magnesium rich phyllosilicates such as nontronite that results from the hydrothermal alteration of a mafic or ultramafic oceanic crust. In the present paper, we study the adsorption of nucleotides on montmorillonite and nontronite, at various pH and ionic strength conditions plausible for Archean sea-water. A thorough characterization of the mineral surfaces shows that nucleotide adsorb mainly on the edge faces of the smectites by ligand exchange between the phosphate groups of the nucleotides and the –OH groups from the edge sites over a wide pH range (4–10). Nontronite is more reactive than montmorillonite. At low pH, additional ion exchange may play a role as the nucleotides become positively charged.

Problem‐based test: Replication of mitochondrial DNA during the cell cycle

Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids, re-replication block, cell fractionation, Svedberg (sedimentation constant = [ S]), nuclear DNA, mitochondrial DNA, heavy and light mitochondrial DNA chains, heteroplasmy, mitochondrial diseases

Non-enzymatic Polymerization of Nucleic Acids from Monomers: Monomer Self- Condensation and Template-Directed Reactions

This review deals with the state-of-the-art techniques in non-enzymatic nucleic acid condensation from monomers. In particular, the procedures called monomer self-condensation and template-directed monomer condensation are described, which have been developed to achieve efficient synthesis of long nucleic acid polymers or to sequence-specifically amplify nucleic acid polymers, respectively. Starting from molecular requirements, details of the polymerization mechanisms and strategies are first presented and then compared. Finally, we discuss the relevance of these strategies to the investigation of possible early molecular information systems on the prebiotic earth and the development of novel synthetic methodologies for nucleic acids. Keywords: Activated nucleotides, micro-environments, monomer self-condensation, non-enzymatic, nucleic acids, synthetic biology, template- directed polymerization, template-directed replication.

Uridine insertion/deletion editing in trypanosomes: a playground for RNA‐guided information transfer

RNA editing is a collective term referring to enzymatic processes that change RNA sequence apart from splicing, 5' capping or 3' extension. In this article, we focus on uridine insertion/deletion mRNA editing found exclusively in mitochondria of kinetoplastid protists. This type of editing corrects frameshifts, introduces start and stops codons, and often adds much of the coding sequence to create an open reading frame. The mitochondrial genome of trypanosomatids, the most extensively studied clade within the order Kinetoplastida, is composed of ∼50 maxicircles with limited coding capacity and thousands of minicircles. To produce functional mRNAs, a multitude of nuclear-encoded factors mediate interactions of maxicircle-encoded pre-mRNAs with a vast repertoire of minicircle-encoded guide RNAs. Editing reactions of mRNA cleavage, U-insertions or U-deletions, and ligation are catalyzed by the RNA editing core complex (RECC, the 20S editosome) while each step of this enzymatic cascade is directed by guide RNAs. These 50-60 nucleotide (nt) molecules are 3' uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Remarkably, the information transfer between maxicircle and minicircle transcriptomes does not rely on template-dependent polymerization of nucleic acids. Instead, intrinsic substrate specificities of key enzymes are largely responsible for the fidelity of editing. Conversely, the efficiency of editing is enhanced by assembling enzymes and RNA binding proteins into stable multiprotein complexes. WIREs RNA 2011 2 669-685 DOI: 10.1002/wrna.82 For further resources related to this article, please visit the WIREs website.

In Silico Study of the Inhibition of Taq Polymerase by Fullerol C60(oh)20

Polymerases are enzyme proteins catalyzing the polymerization of nucleic acids by the addition of nucleotides to a substrate ss-DNA (or RNA) against a template ss-DNA, making the correct Watson-Crick base pairs. TaqDNA polymerase (taqpol), a thermostable polymerase from the bacterium thermus aquaticus, is widely used in polymerase chain reactions (PCR) - a popular technique for the amplification of a DNA sample. Recent studies have reported that fullerol C60(OH)n, a water-soluabe fullerene derivative, can inhibit PCR [1, 2]. The experimental evidence suggests that fullerol inhibits PCR by interacting with the enzyme taq polymerase [2]. Since polymerases in all organisms use the same mechanism for the polymerization of nucleotides, fullerol may inhibit other polymerases and thus affect DNA duplication in cells. We have used molecular dynamics (MD) and molecular docking to understand the mechanism of taq polymerase inhibition. The crystal structure of polymerizing domain (Klentaq) of the taq polymerase is obtained from [3]. Molecular docking is used to find the binding sites of fullerol on Klentaq (Autodock 4.2 [4]). MD simulation are started from the conformations obtained from docking studies. By a detailed comparison of the simulations of the protein in the presence of fullerol with simulations in the absence of fullerol, insight into the mechanism of inhibition is obtained. [1] X. Meng et. al, J. of Enzyme Inhib. Med. Chem. 22, 293 (2007) [2] J. Shang et. al, Nanotechnology 20, 415101 (2009) [3] Y. Kim et. al, Nature 376, 612 (1995) [4] G. M. Morris et. al, J. Comput. Chem., in press (2009)

Light activation of transcription: photocaging of nucleotides for control over RNA polymerization

We describe the use of ATP caged with [7-(diethylamino)coumarin-4-yl]methyl (DEACM) for light-controlled in vitro transcription reactions. Polymerization is blocked when DEACM is bonded to the gamma phosphate group of the ATP molecule. Controlled light irradiation releases ATP and transcription is initiated. In order to provide full control over the process, conditions involved in substrate release, nucleotide availability after release and the effect of the released coumarin in RNA polymerization were assessed in further detail. Together, our data provide the first direct evidence of control over enzymatic polymerization of nucleic acids through light. This approach may provide researchers with a unique tool for the study of biological processes at a molecular level.

Presidential Address to the Mineralogical Society of America, Salt Lake City, October 18, 2005: Mineral surfaces and the prebiotic selection and organization of biomolecules

One of the most enigmatic steps in Earth.s ancient transition from a lifeless planet to a living world was the process or processes by which prebiotic organic molecules were selected, concentrated, and organized into the essential macromolecules of life. More than a half-century of theory and experiment points to the critical roles of mineral surfaces in the assembly of proteins, lipid bilayers, and genetic polymers. This review considers three aspects of this problem: (1) the self-assembly of lipids, which may be enhanced in the presence of minerals; (2) the role of minerals in polymerization of amino acids and nucleic acids; and (3) the selective adsorption of organic species, including chiral molecules, onto mineral surfaces.

Synthesis and Polymerization of Lipophilic Peptide Nucleic Acids Derived from Stearic and Pentacosa‐10,12‐diynoic Acid.

Synthesis and Polymerization of Lipophilic Peptide Nucleic Acids Derived from Stearic and Pentacosa‐10,12‐diynoic Acid.

Temperature of acrylamide polymerization and electrophoretic mobilities of nucleic acids

The electrophoretic mobilities of DNA, ribosomal RNAs, and pulse-labeled RNAs were compared on polyacrylamide gels polymerized at temperatures from 4 to 35°C and subjected to electrophoresis at a fixed temperature. DNA migrated the same distance irrespective of polymerization temperature, the ribosomal RNAs, and the major pulse-labeled species (a putative rRNA precursor) migrated more rapidly in gels polymerized at higher temperatures. The linearity of the migration versus the log of the molecular weight remained for the five rRNA species used, but the extrapolated molecular weight of the putative precursor ranged from 1.8 × 10 6 to 2.5 × 10 6 depending on polymerization temperatures. By varying polymerization temperatures, the optimal resolution of various groups of RNA species can be obtained. The results are explained in terms of polymerization temperature effects on gel structure as well as nucleic acid conformation.

Nucleoside Diphosphokinase of Salmonella typhimurium

Abstract Nucleoside diphosphokinase from Salmonella typhimurium has been purified and characterized. In many respects, the enzyme is similar to those from higher organisms in that it has a broad specificity for both phosphate donor and recipient and that it functions via a ping-pong mechanism. The enzyme activity of cells is very high at all growth rates but decreases as growth rate increases. Evidence is presented that nucleoside diphosphokinase is sufficient to account for the synthesis of all nucleoside triphosphates required for the polymerization of nucleic acids and that it is probably the only enzyme that catalyzes the synthesis of triphosphates from diphosphates and the γ-phosphate of ATP.

Chapter 19 Analysis of Nucleotide Pools in Animal Cells

This chapter discusses the analysis of nucleotide pools in animal cells. Nucleotides act as carriers and reservoirs of chemical energy, they participate as cofactors, activators, inhibitors, and substrates in myriad enzymatic reactions, and they are the precursors for polymerization of nucleic acids. Most of these processes exhibit some type of dependence of rate on the concentration of participating nucleotides, and in some cases the rate may even be controlled by nucleotide concentration. To establish the existence of such control in vivo , it is necessary to measure intracellular nucleotide concentrations, or pools. The chapter presents a model for nucleotide pools. There are four basic pathways by which components may enter or leave the pool—hence the name “four-factor model”, viz., two processes (anabolism and salvage) supply the pool, and two others (catabolism and utilization) drain it. These processes would apply to the thymine nucleotide pool. The turnover rate of a pool is proportional to the fraction of the pool per unit time which is drained by catabolism and utilization. Conditions which change the draining reactions will affect the turnover rate.

Biochemical and physical changes in shaken suspensions of Pasteurella pestis.

Pasteurella pestis, harvested after 24 to 30 hr of growth in a casein hydrolysate medium at 26 C, was resuspended and shaken in 3% lactose-0.1 m phosphate buffer for 4 hr at the same temperature. Certain characteristics of these starved cells were compared with those of control cells. No differences in the amounts of cellular carbohydrate or lipid were detected. The concentrations of the principal free amino acids were greater in the shaken cells, except that they contained no measureable arginine, and the normally large pools of intracellular tricarboxylic acid cycle intermediates were reduced. Greater viable-cell counts resulted with the cells that were shaken in lactose buffer than with the control cells when each was incubated at 5 C for several weeks. However, the reduced viabilities were apparent losses caused by the formation of aggregates of cells. The clumping of cells was caused by the polymerization of extracellular nucleic acids, principally deoxyribonucleic acid, that were excreted by the cells. Cell clumping could be partially prevented by prior shaking of the suspended cells, which removed some of the deleterious material, or by the action of crystalline deoxyribonuclease.