Stoynov S S, Dolapchiev L B
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria.
Int J Biochem Cell Biol. 2001 Feb;33(2):175-80. doi: 10.1016/s1357-2725(00)00078-9.
All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.
在酿酒酵母中，所有已研究的DNA复制起点都包含DNA解旋元件。引入无限制的负超螺旋会导致DNA解旋元件中的两条DNA链解链。为了理解DNA复制的机制，了解DNA最不稳定的区域是否与复制起点重合很重要。通过用蛇毒磷酸二酯酶切割来研究插入pBR322中的来自酿酒酵母的2μm质粒DNA。其单链内切核酸酶活性允许在DNA解旋元件中切割负超螺旋DNA。通过限制酶绘制毒液磷酸二酯酶水解的位点。这项研究表明，酿酒酵母的2μm质粒DNA的解旋仅在复制起点处由于无限制的负超螺旋而发生。
