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 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.

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel.

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.

A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A.

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.

Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.

We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.