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. 2020 Aug 1;2(3):zcaa013. doi: 10.1093/narcan/zcaa013

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© The Author(s) 2020. Published by Oxford University Press on behalf of NAR Cancer.

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 3. — Replication stress activates MMEJ. (A) Upper panel: scheme for repair of I-SceI-induced DSBs via MMEJ in the EJ2-U2OS cell line; lower panel: repair of I-SceI-induced DSBs after replication stress. EJ2-U2OS cells were treated with the indicated doses of HU and VE-821 (ATRi) for 8 h before DSB induction. (B) Upper panel: scheme for repair of pNS plasmid in mammalian cells after transfection; lower panel: repair of linearized plasmid substrate pNS after replication stress. U2OS cells were treated with the indicated doses of HU and VE-821 for 8 h before transfection of pNS; repaired sequences were analyzed according to (30). (C) Upper panel: in vitro MMEJ repair activity assay scheme; lower panel: MMEJ repair activity of XRCC1-FLAG IP. U2OS cells were transfected with XRCC1-FLAG and immunoprecipitated after treatment. Immunoprecipitated XRCC1-FLAG complexes were incubated with linearized plasmid substrate and competent E. coli cells were transformed with repaired product according to (30). Total colony number reflects individual repair events. Western blot of IP bead eluate after incubation is shown.