Takahashi Shunsuke, Motooka Shinya, Usui Tomohiro, Kawasaki Shohei, Miyata Hidefumi, Kurita Hirofumi, Mizuno Takeshi, Matsuura Shun-ichi, Mizuno Akira, Oshige Masahiko, Katsura Shinji
†Department of Environmental Engineering Science, Graduate School of Science and Technology, Gunma University, Gunma 376-8515, Japan.
‡Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
Anal Chem. 2015 Mar 17;87(6):3490-7. doi: 10.1021/acs.analchem.5b00044. Epub 2015 Mar 4.
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.
在单分子水平上研究了负超螺旋对DNA双螺旋局部变性的影响。通过用包含复制蛋白A的70-kDa亚基的单链DNA结合结构域和增强型黄色荧光蛋白（RPA-YFP）的融合蛋白对单链DNA区域进行染色，然后用YOYO-1对双链DNA区域进行染色，直接观察了λDNA和含有SV40 DNA复制起点（SV40ori-λDNA）的λDNA中的局部变性。在负超螺旋状态下，λDNA和SV40ori-λDNA的局部变性在单链区域表现为单个亮点。当λDNA的负超螺旋密度逐渐增加到0、-0.045和-0.095，以及SV40ori-λDNA的负超螺旋密度逐渐增加到0、-0.047和-0.1时，在λDNA的-0.095和SV40ori-λDNA的-0.1的较高负超螺旋密度下，单链区域的单个亮点频繁出现。然而，对于λDNA和SV40ori-λDNA，分别在低于-0.045和-0.047的负超螺旋密度下，很少观察到单链区域的单个亮点。对于λDNA和SV40ori-λDNA，局部变性发生的概率均随负超螺旋度的增加而增加。
