Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

Takahashi, Shunsuke; Usui, Tomohiro; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

doi:10.1016/j.ab.2014.04.012

Cited by 5 publications

(8 citation statements)

References 30 publications

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“…DOPC liposomes for these modifications were prepared from DOPC, as described in our previous report. 28 Restriction enzymes Dra I, EcoR I, Hinc II, KpnI, and Hind III were purchased from TaKaRa (Shiga, Japan). E. coli NM514 was used as a host for the bacteriophage λgt10 vector DNA (Stratagene, La Jolla, Ca, USA).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Figure 2A illustrates the design of the microflow channel and was prepared as described in our previous reports. 25,26,28 Poly ether ether ketone tubing (I.D. 100 μm, O.D.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The method to modify the surface of the microflow channel with neutravidin and DOPC liposomes was described in our previous report. 28 Microflow channel inlets were connected to a syringe with a syringe pump. For the following experiments, the flow rate was adjusted to 50 μL/h unless otherwise stated.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

et al. 2015

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Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Figure 2A illustrates the design of the microflow channel and was prepared as described in our previous reports. 25,26,28 Poly ether ether ketone tubing (I.D. 100 μm, O.D.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

et al. 2015

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“…The exonuclease activity functions to rectify the incorrect pairing of bases during DNA synthesis [170]. To analyze the dynamic DNA digestion by exonuclease, it is significantly effective to directly observe it at the single-molecule level [56,[171][172][173][174]. Thus, by combining DNA manipulation and single-molecule imaging, the elementary process of DNA digestion by λ exonuclease [56,171], exonuclease III [172,173], and T7 exonuclease [174] has been successfully captured.…”

Section: Single-molecule Imaging For Dna Digestion By Exonucleasementioning

confidence: 99%

“…In the direct observation of DNA digestion by T7 exonuclease, the activity of T7 exonuclease was analyzed on the basis of the length of the single DNA molecule stretched by fluid flow [174]. The single-stranded and double-stranded regions of the single DNA molecule were stained with two different fluorescent dyes.…”

Section: Single-molecule Imaging For Dna Digestion By Exonucleasementioning

confidence: 99%

DNA Manipulation and Single-Molecule Imaging

2021

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DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

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Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

Schreiber,

Prange,

Schäfer

et al. 2024

Molecular Plant

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Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

Cited by 5 publications

References 30 publications

Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

DNA Manipulation and Single-Molecule Imaging

Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

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