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. 2021 Dec 30;50(2):899–914. doi: 10.1093/nar/gkab1253

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — The Mug20-LacI fusion protein lacks DSB hotspot competition at the ade6-3101 hotspot but retains competition at the endogenous mbs1 hotspot. rad50S strains were induced for meiosis, and DNA analyzed as in Figure 2. In each panel, quantification is based on n blots from two independent inductions; error bars indicate SEM (range for n = 2). (A) ade6-3049 but not ade6-3101 plus Mug20-LacI (mug20-231) competes with close hotspots; ade6-M375 is a non-hotspot control. DNA was digested with PmeI and analyzed on three to five blots with a probe at the right end of the 74.2 kb fragment containing ade6. DSBs at sites 1 and 2 were significantly less frequent with ade6-3049 than with ade6-M375 (***P < 0.0001 for site 1 and **P = 0.0004 for site 2) but were not less frequent with the ade6-3101 hotspot (thick black arrow, ∼20 kb). Rather, DSBs at site 1 were moderately stimulated by ade6-3101 plus Mug20-LacI (mug20-231) versus ade6-3101 alone or M375: *P = 0.011 or **0.004, respectively). DSBs at site 2 were not significantly different (P = 0.18 or 0.054, respectively). (B) ade6-3049 but not the ade6-3101 hotspot (DSBs indicated by thick black arrow, ∼110 kb) competes with a distant hotspot. DNA was digested with SacII and analyzed on three or four blots with a probe at the right end of the 150.5 kb fragment with ade6. Only ade6-3049 competed with the strong DSB hotspot 75 kb to the right of ade6 (75R) (*P = 0.015) or with weaker DSBs in between. (C) Mug20-LacI manifests DSB competition on another chromosome. DNA was digested with NotI and analyzed on four to seven blots with a probe at the left end of the 501 kb NotI fragment J. mbs1 was competed by the artificial hotspot ura1::hph in both Mug20 and Mug20-LacI strains (***P < 0.0001). mbs2, an endogenous hotspot 100 kb to the left of mbs1, was also competed by ura1::hph in Mug20-LacI strains (***P < 0.0001). DSBs at mbs3, 200 kb to the right of mbs1, did not differ significantly in these strains. Strains with no black bar (first and third from the left) are ura1⁺.