HeLa细胞核的一种强大连环化活性的表征

Characterization of a potent catenation activity of HeLa cell nuclei.

作者信息

Holden J A, Low R L

出版信息

J Biol Chem. 1985 Nov 25;260(27):14491-7.

Abstract

Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles.

摘要

利用一种检测超螺旋DNA模板连环化的实验方法，我们对在HeLa细胞核粗提物中鉴定出的一种强活性进行了表征和定量分析。连环化反应需要Mg-ATP和一种DNA凝聚剂聚乙烯醇。可使用滤膜结合或琼脂糖凝胶实验来定量活性。在该反应中，DNA拓扑异构酶I使输入的超螺旋DNA松弛，从而提供DNA拓扑异构酶II，这是一种极有利于连环化的模板。DNA拓扑异构酶II优先使松弛的DNA连环化，其对超螺旋DNA的连环化效率要高出100倍。在30℃时，一个DNA拓扑异构酶II分子每分钟能够使约20个松弛的DNA环连环化，但在30℃时，每分钟只能使0.16个超螺旋DNA环连环化。纯化的HeLa拓扑异构酶I在这些实验条件下也能使DNA连环化，但不依赖于ATP。其效率远低于拓扑异构酶II；在30℃时，一个拓扑异构酶I分子每分钟只能使含有5%带切口环的DNA模板中的约3.8×10⁻³个超螺旋DNA分子连环化。这两种酶之间的显著差异使得能够在存在过量拓扑异构酶I的情况下对DNA拓扑异构酶II的活性进行定量分析。与大肠杆菌拓扑异构酶I（ω）不同，HeLa拓扑异构酶I的连环化不受带缺口环的刺激。