Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an “alien” impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.
CITATION STYLE
Larsen, N. B., Hickson, I. D., & Mankouri, H. W. (2018). A molecular toolbox to engineer site-specific DNA replication perturbation. In Methods in Molecular Biology (Vol. 1672, pp. 295–309). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7306-4_20
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