Rodríguez-Campos A
Departamento de Biología Molecular y Celular, Centro de Investigación y Desarrollo, CSIC, Jordi Girona, 18 E-08034, Barcelona, Spain.
J Biol Chem. 1996 Jun 14;271(24):14150-5. doi: 10.1074/jbc.271.24.14150.
Topological knots can be formed in vitro by incubating covalently closed double stranded DNA and purified topoisomerase II from the yeast Saccharomyces cerevisiae in an ATP-dependent reaction. Knotting production requires a starting enzyme/DNA mass ratio of 1. Analysis of knotted DNA was carried out by using both one- and two-dimensional agarose gel electrophoresis. The knots generated are efficiently untied, and give relaxed DNA rings, by catalytic amounts of topoisomerase II, but not by topoisomerase I. Time course analysis shows the knotting formation over relaxed and supercoiled DNA. When supercoiled DNA was used as a susbtrate, knots appear immediately whereas no transient relaxed rings were observed. The cell-free extract from Xenopus oocytes S-150 cannot assemble nucleosomes on knotted DNA templates as revealed by topological and micrococcal nuclease analysis. Nevertheless, the presence of knotted DNA templates does not inhibit the assembly over the relaxed plasmid. Finally, a pretreatment of knotted DNA with trace amounts of topoisomerase II before the addition of the S-150 yields a canonical minichromosome assembled in vitro. Taking into account these results, I suggest a mechanism of chromatin assembly regulation directed by topoisomerase II.
通过在ATP依赖的反应中孵育共价闭合的双链DNA和从酿酒酵母中纯化的拓扑异构酶II，可在体外形成拓扑结。形成结需要起始酶/DNA质量比为1。通过一维和二维琼脂糖凝胶电泳对打结的DNA进行分析。产生的结可被催化量的拓扑异构酶II有效解开，形成松弛的DNA环，但拓扑异构酶I不能解开。时间进程分析显示了在松弛和超螺旋DNA上形成结的过程。当使用超螺旋DNA作为底物时，结立即出现，而未观察到瞬时松弛环。拓扑和微球菌核酸酶分析表明，非洲爪蟾卵母细胞S-150的无细胞提取物不能在打结的DNA模板上组装核小体。然而，打结的DNA模板的存在并不抑制在松弛质粒上的组装。最后，在添加S-150之前，用微量拓扑异构酶II对打结的DNA进行预处理，可产生体外组装的典型微型染色体。考虑到这些结果，我提出了一种由拓扑异构酶II指导的染色质组装调控机制。
