Liu L F, Rowe T C, Yang L, Tewey K M, Chen G L
J Biol Chem. 1983 Dec 25;258(24):15365-70.
Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.
利用P4解结试验，已从多种哺乳动物细胞中纯化出DNA拓扑异构酶II。与原核DNA促旋酶相似，哺乳动物DNA拓扑异构酶II可切割双链DNA，并以共价蛋白质-DNA复合物的形式被捕获。这种切割反应需要对拓扑异构酶II-DNA复合物进行蛋白质变性剂处理，并且在盐和温度方面是可逆的。切割反应逆转后的产物仍然是超螺旋的，这表明假定断裂的DNA的两端被拓扑异构酶紧密地固定着。或者，酶与DNA的相互作用是非共价的，拓扑异构酶与DNA的共价连接是由蛋白质变性剂诱导的。对切割产物的详细表征表明，拓扑异构酶II以四个碱基的交错方式切割DNA，并与每条断裂DNA链突出的5'-磷酸末端共价连接。小牛胸腺DNA拓扑异构酶II在多个特定位点切割SV40 DNA。然而，通过直接核苷酸测序研究确定，在切割位点之间未发现序列同源性。
