Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

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 lambda DNA and lambda DNA containing the SV40 origin of DNA replication (SV40ori-lambda DNA) 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 lambda DNA and SV40ori-lambda DNA 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 lambda DNA and 0, -0.047, and -0.1 for SV40ori-lambda DNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for lambda DNA and -0.1 for SV40ori-lambda DNA. However, single bright spots of the Single-Stranded regions were rarely observed below a negative supercoil density of -0.045 and 0.047 for lambda DNA and SV40ori-lambda DNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both lambda DNA and SV40ori-lambda DNA.