Genetic variant research in hypertension has identified numerous associations but is unlikely to yield new treatments, emphasizing the gap between scientific discovery and clinical application.
Universitat Autonoma de Barcelona (UAB) researchers have produced the most complete map of the bacterial essential interactome, i.e. how proteins combine and interact to perform functions essential for their survival.
DNA replication in cells occurs on crowded and often damaged template DNA, forming potentially deleterious roadblocks to the progressing replication fork. Numerous tools have been developed to investigate the mechanisms of DNA replication and the fate of stalled replication forks. Here, we describe single-molecule fluorescence imaging methods to visualize processive DNA replication and replication fork stalling at site-specific nucleoprotein complexes. Using dCas9 as a protein barrier and rolling-circle DNA templates, we visualize effective, stable, and site-specific blocking of the Escherichia coli replisome. Additionally, we present a protocol to produce an 18-kb rolling-circle DNA template that provides increased spatial resolution in imaging the interplay between replisomes and roadblocks. These methods can be used to investigate encounters of the replisome with nucleoprotein complexes at the single-molecule level, providing important mechanistic details of replisome stalling and d