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

Figure 2.

Figure 2.

Linear element fusion protein Mug20-LacI induces abundant DSBs at the ade6-3101 hotspot. (A) Formation and accumulation of DSBs in rad50S strains with ade6-3101 containing 8 lacO operators (at the thick black arrow) alone, with Mug20-LacI alone (mug20-231), or with both to generate the ade6-3101 hotspot. ade6-3049 (thick white arrow) is a non-LacI-lacO DSB hotspot control. Bracket indicates the ∼2 kb region of DSB formation in the two strains. Cells were induced for meiosis and harvested at the indicated times. DNA was digested with BsrGI and analyzed by electrophoresis and Southern blot hybridization using a probe at the right end of the 11.8 kb fragment with ade6. Black ovals on the left margin indicate a DNA ladder (1 kb Plus, Invitrogen; from the top 15, 10, 8, 7, 6, 5, 4, and 3 kb). Quantification is based on 2 or 3 blots from two independent inductions; error bars indicate the range or SEM. (B) Early formation and timely repair of DSBs in a rad50+ strain with ade6-3101 and Mug20-LacI (mug20-231). DNA was analyzed as in (A) after digestion with PmeI using a probe at the right end of the 74.2 kb fragment with ade6. DSBs at ade6-3101 are indicated by the thick black arrow (∼20 kb fragment); endogenous DSB sites 1 and 2 are 15 and 5 kb from ade6. Quantification is based on two independent inductions; error bars (some invisible) indicate the range. Note that DSBs at ade6-3101 are visible before replication is complete at 3 h, but DSBs at endogenous site 1, site 2, and mbs1 are not (see also Supplementary Figure S1).