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. 2018 Apr 6;46(10):5075–5096. doi: 10.1093/nar/gky254

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© The Author(s) 2018. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 8. — rad1R218A function in SSA. (A) rad1R218A-3HA was integrated into the chromosome, replacing endogenous RAD1. To determine expression levels, α-HA antibody was used to immunoprecipitate HA-tagged protein from cell lysates grown to mid-log phase for rad1R218A-3HA, RAD1–3HA, and rad1Δ w. A western blot of eluates was performed using α−HA antibody. rad1Δ serves as a negative control. An arrow indicates the Rad1–3HA or rad1R218A-3HA band. (B) Cartoon of SSA in the EAY1141 (short ∼250 bp ura3 repeats) and YMV80 (long ∼ 1.3 kb leu2 repeats) strains used to assess 3′ NHTR.(C) Msh2–Msh3-dependent SSA assays with short repeats were performed. D. Msh2–Msh3-independent SSA assays with long repeats were performed. Percent survival (induced/uninduced) was calculated by determining the viability of cells after a 5 hour induction of HO expression. Data represents the mean ± SEM of at least six independent experiments with at least two independent isolates. The following strains were used: RAD1 (black), rad1Δ (dark red), rad1D825A (orange), rad1R218A (teal) and msh2Δ (magenta).