Developing an in vitro Next Generation Sequencing Based Approach to Map Single-Stranded DNA cleavage by Topoisomerases

Authors

• NI Dmitrieva
• D Liu
• CO Wu
• DR Rosing
• M Boehm

Abstract

Topoisomerases are enzymes critical to cell survival that regulate the topology of DNA. To control DNA topology, they induce transient breaks in the double helix. If these breaks become permanent, they are highly toxic to cells, making topoisomerases a well-established target for anticancer and antimicrobial drugs. There are two functional classes of topoisomerases, Type I and Type II, which are mechanistically differentiated by whether they generate single-stranded breaks (SSBs) or double-stranded breaks (DSBs), respectively. Whereas the magnitude of topoisomerase cleavage has been well-studied, technical limitations have made it difficult to determine the location of cleavage sites with nucleotide resolution. Recently, our lab developed an in vitro technique, Simplified High Accuracy eNd sequencing (SHAN-seq) to elucidate sequence-specific double-stranded cleavage by type II topoisomerases. However, type I topoisomerases and type II topoisomerases (when treated with certain drugs) nick DNA, creating single-stranded breaks, that are not currently captured by SHAN-seq. Hence, we have extended SHAN-seq to capture the location of single-stranded breaks. We validated this method using nicking enzymes with well-defined sites on a plasmid. This work enables the full mapping of the cleavage sequence preferences for both type I and II topoisomerases and how the cleavage location and extent vary with DNA topology and topoisomerase inhibitors, which have important implications for DNA repair and determining the effects of anticancer and antimicrobial drugs.

Scientific Focus Area: Biomedical Engineering and Biophysics

This page was last updated on Monday, September 25, 2023