title: Direct observation method of individual single-stranded DNA molecules using fluorescent replication protein A.
abstract: Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.
authors.affiliation: Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Oshige
authors.forename: Shohei
authors.initials: S
authors.lastname: Kawasaki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takano
authors.forename: Kouji
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Mizuno
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Matsuura
authors.forename: Akira
authors.initials: A
authors.lastname: Mizuno
authors.forename: Shinji
authors.initials: S
authors.lastname: Katsura
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D008722:Methods; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A