Andersen A H, Christiansen K, Zechiedrich E L, Jensen P S, Osheroff N, Westergaard O
Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
Biochemistry. 1989 Jul 25;28(15):6237-44. doi: 10.1021/bi00441a015.
The strand specificity of topoisomerase II mediated DNA cleavage was analyzed at the nucleotide level by characterizing the enzyme's interaction with a strong DNA recognition site. This site was isolated from the promoter region of the extrachromosomal rRNA genes of Tetrahymena thermophila and was recognized by type II topoisomerases from a variety of phylogenetically diverse eukaryotic organisms, including Drosophila, Tetrahymena, and calf thymus. When incubated with this site, topoisomerase II was found to introduce single-stranded breaks (i.e., nicks) in addition to double-stranded breaks in the nucleic acid backbone. Although the nucleotide position of cleavage on both the noncoding and coding strands of the rDNA remained unchanged, the relative ratios of single- and double-stranded DNA breaks could be varied by altering reaction conditions. Under all conditions which promoted topoisomerase II mediated DNA nicking, the enzyme displayed a 3-10-fold specificity for cleavage at the noncoding strand of its recognition site. To determine whether this specificity of topoisomerase II was due to a faster forward rate of cleavage of the noncoding strand or a slower rate of its religation, a DNA religation assay was performed. Results indicated that both the noncoding and coding strands were religated by the enzyme at approximately the same rate. Therefore, the DNA strand preference of topoisomerase II appears to be embodied in the enzyme's forward cleavage reaction.
通过表征拓扑异构酶II与一个强DNA识别位点的相互作用，在核苷酸水平上分析了拓扑异构酶II介导的DNA切割的链特异性。该位点是从嗜热四膜虫的染色体外rRNA基因的启动子区域分离出来的，并且被来自多种系统发育上不同的真核生物（包括果蝇、四膜虫和小牛胸腺）的II型拓扑异构酶所识别。当与该位点一起孵育时，发现拓扑异构酶II除了在核酸主链中引入双链断裂外，还会引入单链断裂（即切口）。尽管rDNA的非编码链和编码链上切割的核苷酸位置保持不变，但通过改变反应条件可以改变单链和双链DNA断裂的相对比例。在所有促进拓扑异构酶II介导的DNA切口形成的条件下，该酶在其识别位点的非编码链上的切割显示出3至10倍的特异性。为了确定拓扑异构酶II的这种特异性是由于非编码链的切割前向速率更快还是其重新连接速率更慢，进行了DNA重新连接试验。结果表明，非编码链和编码链被该酶重新连接的速率大致相同。因此，拓扑异构酶II对DNA链的偏好似乎体现在该酶的前向切割反应中。
