title: A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A.
abstract: Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan. t12801416@gunma-u.ac.jp.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan. t09801411@gunma-u.ac.jp.
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan. b.history12@gmail.com.
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 4418580, Japan. kurita@ens.tut.ac.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurita
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Saitama 3510198, Japan. tmizuno@riken.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mizuno
authors.affiliation: Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 9838551, Japan. matsuura-shunichi@aist.go.jp.
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 4418580, Japan. mizuno@ens.tut.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Mizuno
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan. oshige@cee.gunma-u.ac.jp.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan. t09801411@gunma-u.ac.jp.
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D005453:Fluorescence; D008164:Luminescent Proteins / Q000378:metabolism*; D044085:Microfluidics / Q000379:methods*; D051716:Replication Protein A / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D051716:Replication Protein A; C054562:yellow fluorescent protein, Bacteria; D004256:DNA Polymerase I