II 型拓扑异构酶 (topos) 是一类普遍存在的重要酶，可在 DNA 上形成短暂的酶结合双链断裂，称为切割复合物。这些裂解复合物在 DNA 上的位置和频率对于细胞功能、基因组稳定性以及许多临床上重要的抗癌和抗菌药物（例如抗生素）非常重要。喹诺酮类药物。我们开发了一种简单的高精度末端测序 (SHAN-seq) 方法，可在体外灵敏地绘制 DNA 上的 II 型拓扑切割复合物。使用 SHAN-seq，我们在超螺旋 pBR322 DNA 的数百个位点（大约每 10 bp 一个位点）检测到了大肠杆菌旋转酶和拓扑异构酶 IV 裂解复合物，频率相差两到三个数量级。这些站点包括以前确定的站点和 20-50 倍的新站点。我们表明，这些位点的裂解复合物的位置和频率是酶特异性的，并且在喹诺酮、环丙沙星存在的情况下显着变化，但不随DNA超螺旋手性（即负超螺旋与正超螺旋）变化。 SHAN-seq 精湛的灵敏度为 DNA 上旋转酶和拓扑异构酶 IV 切割复合物的分布提供了前所未有的单核苷酸分辨率视图。此外，这些酶可以比先前已知的相对较少的位点以更多数量级的位点切割 DNA，这一发现解决了这些酶如何解决整个基因组拓扑问题的明显悖论。 由牛津大学出版社代表核酸研究出版 2024 。

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.Published by Oxford University Press on behalf of Nucleic Acids Research 2024.