Morgan Ian L, McKie Shannon J, Kim Rachel, Seol Yeonee, Xu Jing, Harami Gabor, Maxwell Anthony, Neuman Keir C
biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.
bioRxiv. 2024 May 17:2024.05.17.594727. doi: 10.1101/2024.05.17.594727.
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 . Using SHAN-seq, we detected 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 vs. 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.
II型拓扑异构酶是一类普遍存在且至关重要的酶，它们在DNA上形成与酶短暂结合的双链断裂，称为切割复合体。这些切割复合体在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（如喹诺酮类）而言都很重要。我们开发了一种简单的高精度末端测序（SHAN-seq）方法，用于灵敏地绘制DNA上II型拓扑异构酶的切割复合体图谱。使用SHAN-seq，我们在超螺旋pBR322 DNA上的数百个位点检测到了促旋酶和拓扑异构酶IV的切割复合体，大约每十个碱基对有一个位点，频率变化范围达两到三个数量级。这些位点包括先前已鉴定的位点以及数量多出20至50倍的新位点。我们发现，这些位点处切割复合体的位置和频率具有酶特异性，在喹诺酮类药物环丙沙星存在的情况下会有显著变化，但不受DNA超螺旋手性（即负超螺旋与正超螺旋）的影响。SHAN-seq极高的灵敏度提供了关于促旋酶和拓扑异构酶IV切割复合体在DNA上分布的前所未有的单核苷酸分辨率视图。此外，这些酶能够在比先前已知的相对较少位点数量多几个数量级的位点切割DNA这一发现，解决了这些酶如何在整个基因组中解决拓扑问题这一明显的矛盾。
