A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation
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.
Key wordsReplication fork barrier DNA replication stress Tus-Ter
Work in the authors’ laboratory is funded by the Danish National Research Foundation (DNRF115), The European Research Council, The Novo Nordisk Foundation, and The Nordea Foundation.
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