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. 2018:1672:295-309.
doi: 10.1007/978-1-4939-7306-4_20.

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

Nicolai B Larsen  1 , Ian D Hickson  1 , Hocine W Mankouri  2
Affiliations

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

Nicolai B Larsen et al. Methods Mol Biol. 2018.

Abstract

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.

Keywords: DNA replication stress; Replication fork barrier; Tus-Ter.

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