AI Article Synopsis
- DNA replication happens on crowded and often damaged DNA, leading to potential issues for the replication process.
- Researchers have developed single-molecule fluorescence imaging techniques to study DNA replication and stalled replication forks in detail.
- Using the dCas9 protein to create specific barriers, they visualized how the E. coli replisome interacts with these blockages, enhancing understanding of how replisomes stall and how they can be rescued or restarted.
Article Abstract
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 downstream rescue or restart pathways.
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| http://dx.doi.org/10.1007/978-1-0716-3377-9_11 | DOI Listing |
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