Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state

<p>Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.</p

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

Distribution and metabolism of ¹⁴C-sulfoquinovosylacylpropanediol (¹⁴C-SQAP) after a single intravenous administration in tumor-bearing mice

<p></p><p>Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65.</p><p>For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-¹⁴C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body.</p><p>The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-¹⁴C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice.</p><p>The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney.</p><p>Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.</p><p></p> <p>Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65.</p> <p>For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-¹⁴C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body.</p> <p>The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-¹⁴C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice.</p> <p>The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney.</p> <p>Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.</p