title: Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.
abstract: Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.
authors.affiliation: a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.|f Japan Society for the Promotion of Science.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Motooka
authors.affiliation: a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.affiliation: b Department of Environmental and Life Sciences, Graduate School of Engineering , Toyohashi University of Technology , Toyohashi , Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurita
authors.affiliation: c Cellular Dynamics Laboratory , RIKEN, Wako , Saitama , Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mizuno
authors.affiliation: d Research Institute for Chemical Process Technology , National Institute of Advanced Industrial Science and Technology (AIST) , Sendai , Japan.
authors.forename: Shun-Ichi
authors.initials: SI
authors.lastname: Matsuura
authors.affiliation: e Faculty of Science, Institute for Biomolecular Science , Gakushuin University , Tokyo , Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.affiliation: b Department of Environmental and Life Sciences, Graduate School of Engineering , Toyohashi University of Technology , Toyohashi , Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Mizuno
authors.affiliation: a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.affiliation: a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008280:Magnetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D052898:Optical Tweezers; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004278:DNA, Superhelical; D004279:DNA, Viral
keywords: DNA unwinding; SV40 large tumor antigen; initiation of DNA replication; negative supercoil; single-molecule observation