A quest for coordination among activities at the replisome

2019 ◽  
Vol 47 (4) ◽  
pp. 1067-1075 ◽  
Author(s):  
Nitin Kapadia ◽  
Rodrigo Reyes-Lamothe
Keyword(s):  
Single Molecule ◽  
Genetic Material ◽  
Model Organisms ◽  
Valuable Insight ◽  
Double Stranded Dna ◽  
In Vitro Reconstitution ◽  
Dna Chain ◽  
Protein Ensembles

Abstract Faithful DNA replication is required for transmission of the genetic material across generations. The basic mechanisms underlying this process are shared among all organisms: progressive unwinding of the long double-stranded DNA; synthesis of RNA primers; and synthesis of a new DNA chain. These activities are invariably performed by a multi-component machine called the replisome. A detailed description of this molecular machine has been achieved in prokaryotes and phages, with the replication processes in eukaryotes being comparatively less known. However, recent breakthroughs in the in vitro reconstitution of eukaryotic replisomes have resulted in valuable insight into their functions and mechanisms. In conjunction with the developments in eukaryotic replication, an emerging overall view of replisomes as dynamic protein ensembles is coming into fruition. The purpose of this review is to provide an overview of the recent insights into the dynamic nature of the bacterial replisome, revealed through single-molecule techniques, and to describe some aspects of the eukaryotic replisome under this framework. We primarily focus on Escherichia coli and Saccharomyces cerevisiae (budding yeast), since a significant amount of literature is available for these two model organisms. We end with a description of the methods of live-cell fluorescence microscopy for the characterization of replisome dynamics.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

scholarly journals Two Decades of Triazine Dendrimers

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4774
Author(s):  
Eric E. Simanek
Keyword(s):  
Genetic Material ◽  
Structural Complexity ◽  
Bioactive Molecules ◽  
Bioactive Materials ◽  
In Vivo Models ◽  
Tumor Subtypes ◽  
Gas Streams

For two decades, methods for the synthesis and characterization of dendrimers based on [1,3,5]-triazine have been advanced by the group. Motivated by the desire to generate structural complexity on the periphery, initial efforts focused on convergent syntheses, which yielded pure materials to generation three. To obtain larger generations of dendrimers, divergent strategies were pursued using iterative reactions of monomers, sequential additions of triazine and diamines, and ultimately, macromonomers. Strategies for the incorporation of bioactive molecules using non-covalent and covalent strategies have been explored. These bioactive materials included small molecule drugs, peptides, and genetic material. In some cases, these constructs were examined in both in vitro and in vivo models with a focus on targeting prostate tumor subtypes with paclitaxel conjugates. In the materials realm, the use of triazine dendrimers anchored on solid surfaces including smectite clay, silica, mesoporous alumina, polystyrene, and others was explored for the separation of volatile organics from gas streams or the sequestration of atrazine from solution. The combination of these organics with metal nanoparticles has been probed. The goal of this review is to summarize these efforts.

scholarly journals Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

2019 ◽  
Vol 476 (10) ◽  
pp. 1521-1537 ◽  
Author(s):  
Emma J. Goldberg ◽  
Katherine A. Buddo ◽  
Kelsey L. McLaughlin ◽  
Regina F. Fernandez ◽  
Andrea S. Pereyra ◽  
...  
Keyword(s):  
Acid Oxidation ◽  
Valuable Insight ◽  
Keto Acid ◽  
Isolated Mitochondria ◽  
Mouse Tissues ◽  
Branched Chain

Abstract Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

2020 ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

scholarly journals Cytokinesis in Eukaryotes

2002 ◽  
Vol 66 (2) ◽  
pp. 155-178 ◽  
Author(s):  
David A. Guertin ◽  
Susanne Trautmann ◽  
Dannel McCollum
Keyword(s):  
Genetic Model ◽  
Plant Cells ◽  
Complex Problem ◽  
Model Organisms ◽  
Animal Cells ◽  
Contractile Ring ◽  
Daughter Cells ◽  
Intense Research

SUMMARY Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells. Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches. In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis. While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms. Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex. As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different. However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells. In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing. In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research. In conclusion, we discuss how certain cells have utilized cytokinesis to direct development.

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