title: Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.
abstract: 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.
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Motooka
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Usui
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, 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: §Cellular Dynamics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
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 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 Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.affiliation: †Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D010582:Bacteriophage lambda; D004278:DNA, Superhelical / Q000737:chemistry*; D008958:Models, Molecular; D009691:Nucleic Acid Denaturation; D013997:Time Factors
chemical_list: D004278:DNA, Superhelical