סמינר לפיזיקה של מערכות ביולוגיות וחומרים רכים: Visualizing DNA topology regulation at the single-molecule level
Dr. Graeme King, University College London, UK
The topological state of DNA in vivo is constantly in flux. Genomic processes such as replication and recombination often lead to the formation of entangled DNA structures. Furthermore, local regions of underwound or overwound DNA (supercoiled DNA) frequently occur, which can influence the binding of proteins such as transcription factors and architectural proteins. Here, I will discuss two new single-molecule assays, based on combined optical tweezers and fluorescence microscopy, that have enabled direct observation of DNA topology regulation at the single-molecule level. First, I will describe recent work that has shed new light on how the human topoisomerase enzyme TopoIIIa can resolve entangled DNA structures and how its mechanism can be altered by the presence of co-factors. Second, I will present a novel assay to generate negatively supercoiled (underwound) DNA using optical tweezers and demonstrate how we have applied this to study the mechanism of supercoil relaxation by TopoIIIa and also probe the dynamics of transcription factor binding to supercoiled DNA. Together, these findings advance our understanding of how the cell regulates DNA topology and how DNA topology, in turn, influences DNA-protein interactions.