Biochemical and biophysical properties of positively supercoiled DNA
| Autor: | Yingting Liu, Zi-Chun Hua, Fenfei Leng, Andrea M Berrido, Yuk-Ching Tse-Dinh |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Circular dichroism Ultraviolet Rays Biophysics Fluorescence spectrometry Nucleic Acid Denaturation Biochemistry DNA gyrase Article 03 medical and health sciences chemistry.chemical_compound Escherichia coli Denaturation (biochemistry) Fluorescent Dyes Electrophoresis Agar Gel 030102 biochemistry & molecular biology biology DNA Superhelical Chemistry Circular Dichroism Topoisomerase Organic Chemistry Intercalating Agents Spectrometry Fluorescence 030104 developmental biology Template DNA Topoisomerases Type I DNA Gyrase biology.protein DNA supercoil DNA Plasmids |
| Zdroj: | Biophysical Chemistry. 230:68-73 |
| ISSN: | 0301-4622 |
| DOI: | 10.1016/j.bpc.2017.08.008 |
| Popis: | In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (−) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (−) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (−) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (−) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (−) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (−) sc DNA. |
| Databáze: | OpenAIRE |
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