Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

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DNA Replication Machinery: Functional Characterization of a Complex Containing DNA Polymerase α, DNA Polymerase δ, and Replication Factor C Suggests an Asymmetric DNA Polymerase Dimer†

Biochemistry ◽

10.1021/bi952455k ◽

1996 ◽

Vol 35 (18) ◽

pp. 5764-5777 ◽

Cited By ~ 31

Author(s):

Giovanni Maga ◽

Ulrich Hübscher

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Functional Characterization ◽

Replication Factor C ◽

Replication Machinery ◽

Replication Factor ◽

Dna Polymerase Δ ◽

Dna Polymerase Α ◽

Factor C

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Functional Characterization of the Genes Coding for the Terminal Protein and DNA Polymerase from Bacteriophage GA-1. Evidence for a Sliding-back Mechanism During Protein-primed GA-1 DNA Replication

Journal of Molecular Biology ◽

10.1006/jmbi.1996.0653 ◽

1996 ◽

Vol 264 (3) ◽

pp. 453-464 ◽

Cited By ~ 36

Author(s):

Belén Illana ◽

Luis Blanco ◽

Margarita Salas

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Functional Characterization ◽

Terminal Protein

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Role of conserved residues in the activity of adenovirus preterminal protein

Journal of General Virology ◽

10.1099/0022-1317-82-8-1917 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1917-1927 ◽

Cited By ~ 6

Author(s):

Catherine H. Botting ◽

Ronald T. Hay

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Insect Cells ◽

Expression System ◽

Baculovirus Expression System ◽

Conserved Residues ◽

Baculovirus Expression ◽

Origin Of Dna Replication ◽

The Individual

Preterminal protein (pTP) is a component of the preinitiation complex which forms at the adenovirus origin of DNA replication and acts as the protein primer during DNA synthesis. In order to determine the role of various regions of the molecule a series of 18 mutations was introduced into conserved motifs of pTP which were predicted to be surface exposed, and the mutants expressed in insect cells using a baculovirus expression system. Their ability to initiate DNA replication was assessed and the effect the mutations have on the individual interactions which contribute to the formation of the pre-initiation complex was determined. Classes of mutants could be identified which were unable to bind DNA or interact with the adenovirus DNA polymerase, but one class of mutants retained these activities and yet failed to initiate DNA replication. These mutants therefore identify regions of pTP required for different aspects of adenovirus DNA replication.

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E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

Scientific Reports ◽

10.1038/s41598-019-51031-0 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Andrea Bogutzki ◽

Natalie Naue ◽

Lidia Litz ◽

Andreas Pich ◽

Ute Curth

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Protein Interactions ◽

Dna Polymerase Iii ◽

Protein Protein Interactions ◽

Single Stranded Dna ◽

E Coli ◽

Polymerase Iii ◽

C Terminus ◽

Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽

10.3390/plants10030524 ◽

2021 ◽

Vol 10 (3) ◽

pp. 524

Author(s):

Bingqi Wu ◽

Zhiting Chen ◽

Xiaohui Xu ◽

Ronghua Chen ◽

Siwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Heterologous Gene Expression ◽

Expression System ◽

Functional Characterization ◽

Transient Gene Expression ◽

High Mobility ◽

Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Scientific Reports ◽

10.1038/s41598-021-94181-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Masuzu Kikuchi ◽

Keiichi Kojima ◽

Shin Nakao ◽

Susumu Yoshizawa ◽

Shiho Kawanishi ◽

...

Keyword(s):

Escherichia Coli ◽

Proton Pump ◽

Expression System ◽

Spectroscopic Characterization ◽

Functional Expression ◽

Proton Pumping ◽

E Coli ◽

Oxyrrhis Marina ◽

Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-021-01854-y ◽

2021 ◽

Author(s):

I. B. Trindade ◽

G. Hernandez ◽

E. Lebègue ◽

F. Barrière ◽

T. Cordeiro ◽

...

Keyword(s):

Functional Characterization ◽

Paramagnetic Nmr ◽

E Coli ◽

The Past ◽

Internal Cavities ◽

Structural Differences ◽

Bond Strengths ◽

Redox Bohr Effect ◽

Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

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