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. 2021 May;31(5):775–788. doi: 10.1101/gr.271155.120

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© 2021 Tripuraneni et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 1. — Inducible DSB at PHO5. (A) Schematic illustrating the ectopic 117-bp HO cut site (HOcs) that was engineered 579 bp upstream of the endogenous PHO5 locus on Chr II (marked in red). The +1 nucleosome of PHO5 is annotated 4L in this illustration. A predicted Sum1 protein binding site is illustrated with a small yellow square. Expression of the HO endonuclease is regulated by an inducible promoter. Nucleosomes are numbered and annotated based on their distance and orientation (left/right) relative to the HO cut site. (B) Southern blot analysis of HO endonuclease-induced cutting near PHO5 with fraction of cut DNA (y-axis) plotted as a function of time (x-axis) for the WT, yku70Δ, and mre11Δ strains. Dotted red line depicts cleavage of 90% of the DNA. (C) Chromatin accessibility surrounding the break greatly increases for WT, yku70Δ, but is limited in mre11Δ strains. Greater time postinduction (min) is plotted in darker shades of blue indicated in the legend (right). (D) Direct comparison between the WT and two mutant strains for the terminal (120 min) MNase sensitivity (in C) which has been smoothed.