Gene 6 Exonuclease of Bacteriophage T7

Kerr, Carol; Sadowski, Paul D.

doi:10.1016/s0021-9258(19)45790-6

Cited by 124 publications

(15 citation statements)

References 20 publications

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“…Several salts were examined for their inhibitory effects, and it was found that KC1, LiCl, and CsCl all inhibited the exonuclease activity to an extent comparable to that caused by NaCl. As previously reported (Kerr & Sadowski, 1972a), 20 mM KC1 has a stimulatory effect (data not shown). Furthermore, the T7 DNA binding protein overcame the inhibitory effects of all the salts tested, producing stimulations that ranged from 8to 20-fold.…”

Section: Resultssupporting

confidence: 90%

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Roberts¹,

Sadowski²,

Wong³

1982

Biochemistry

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Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.

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Section: Resultssupporting

confidence: 90%

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Roberts¹,

Sadowski²,

Wong³

1982

Biochemistry

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“…A single-stranded DNA modified with MB at the 5′-terminus, denoted as MB-DNA, was designed and adopted as the signal probe, which hybridizes with part of the target miRNA sequences from the 3′-end. In the absence of the target miRNA, as illustrated in Scheme a, MB-DNA retains its single-stranded conformation and thus can resist the digestion by T7 Exo, a sequence-independent nuclease that catalyzes the stepwise removal of mononucleotides from the recessed or blunt 5′-termini of double-stranded DNA or RNA-DNA duplex. − Due to the repulsion between the both negatively charged single-stranded MB-DNA and the ITO electrode surface, it is rather difficult for MB-DNA to reach the ITO electrode surface. Upon visible light (λ = 627 nm) illumination, MB can be excited to MB*, and the subsequent oxidation of AA (acting as the electron donor) by MB* to produce leuco-MB and oxidized AA takes place.…”

Section: Resultsmentioning

confidence: 99%

Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay

et al. 2018

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In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time-consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and nonmodified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labeled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labeled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labeled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labeled mononucleotides, significantly increased photocurrent signal is observed for MB-labeled mononucleotides as compared to that of MB-DNAs. Therefore, via this "signal-on" mode and the T7 Exo facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogeneous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness, and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research.

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“…No noticeable enrichment of any macrocycles was observed, possibly as a result of the moderate diversity of the library and the lack of a privileged CA-II-binding structure. [15] When we selected the same library with immobilized CA-II, we also did not observe a significant enrichment of macrocycles (Figure S10). To control for false positives from ExoI resistance, we also performed a selection without CA-II and observed no enrichment (Figure S11); this type of control is analogous to the "beads-only" selection frequently used in immobilizationbased selection.…”

mentioning

confidence: 84%

Selection of DNA‐Encoded Small Molecule Libraries Against Unmodified and Non‐Immobilized Protein Targets

Zhao

Chen

et al. 2014

Angew Chem Int Ed

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The selection of DNA-encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand-induced photo-crosslinking, we show that iterated selections of DNA-encoded libraries can be realized with unmodified and non-immobilized protein targets.

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Gene 6 Exonuclease of Bacteriophage T7

Cited by 124 publications

References 20 publications

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein

Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay

Selection of DNA‐Encoded Small Molecule Libraries Against Unmodified and Non‐Immobilized Protein Targets

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