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. 2019 Oct 1;38(24):e101751. doi: 10.15252/embj.2019101751

Figure 3. BIR/RMD is stimulated by oncogene overexpression and replication fork collapse.

Figure 3

  1. Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP‐SSA‐3427‐3) cell line carrying Tet‐on‐cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 μg/ml), which was added 24 h before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.
  2. ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP‐SSA‐3427‐3) cells infected with lentivirus‐expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 μg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.
  3. A diagram showing the BIR/RMD model when a single‐ended DSB is generated after replication fork collapse.
  4. RMD is stimulated when Cas9‐D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP‐SSA‐3427‐17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9‐D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom).
  5. RMD induced by Cas9‐D10A is dependent on POLD3. U2OS (EGFP‐SSA‐3427‐17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control, and RMD was determined after cleavage by the indicated sgRNAs/Cas9‐D10A. The distance between sgRNA/Cas9‐D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.
  6. p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP‐SSA‐3427‐3) cells carrying the Tet‐on‐cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 μg/ml) added 24 h before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.
Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not significant, two‐tailed non‐paired t‐test.