title: Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel.
abstract: T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Usui
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Miyata
authors.affiliation: Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurita
authors.affiliation: Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 983-8551, Japan.
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Matsuura
authors.affiliation: Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Mizuno
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. Electronic address: katsura@cee.gunma-u.ac.jp.
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C416802:SYTOX Orange dye; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease
keywords: Double-stranded DNA; Single-molecule observation; Single-stranded DNA; T7 Exonuclease