Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State
journal contribution
posted on 2015-03-17, 00:00 authored by Shunsuke Takahashi, Shinya Motooka, Tomohiro Usui, Shohei Kawasaki, Hidefumi Miyata, Hirofumi Kurita, Takeshi Mizuno, Shun-ichi Matsuura, Akira Mizuno, Masahiko Oshige, Shinji KatsuraEffects
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.
