期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：12
页码：7974-7979
DOI：10.1073/pnas.122007999
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The topoisomerase (topo) III enzymes are found in organisms ranging from bacteria to humans, yet the precise cellular function of these enzymes remains to be determined. We previously found that Drosophila topo III{beta} can relax plasmid DNA only if the DNA is first hypernegatively supercoiled. To investigate the possibility that topo III{beta} requires a single-stranded region for its relaxation activity, we formed R-loops and D-loops in plasmids. In addition to containing a single-stranded region, these R-loops and D-loops have the advantage of being covalently closed and supercoiled, thus allowing us to assay for supercoil relaxation. We found that topo III{beta} preferentially cleaves, rather than relaxes, these substrates. The cleavage of the R-loops and D-loops, which is primarily in the form of nicking, occurs to a greater extent at a temperature that is lower than the optimal temperature for relaxation of hypernegatively supercoiled plasmid. In addition, the cleavage can be readily reversed by high salt or high temperature, and the products fail to enter the gel in the absence of proteinase K treatment and are not observed with an active-site Y332F mutant of topo III{beta
