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. 2017 Dec 11;8:2039. doi: 10.1038/s41467-017-02146-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — CDK1/2-dependent phosphorylation promotes RECQL4 participation in repair of DSBs. a–c In vitro phosphorylation of RECQL4 by CDK1/Cyclin A (a), CDK2/Cyclin A (b), and CDK2/Cyclin E (c). 3×FLAG-tagged RECQL4 WT and RQ4-2A mutant were purified from HEK293T cells, and pre-treated with λPP. After washing off λPP, the RECQL4 proteins were incubated with CDK1/2 and their cyclin partners. Phosphorylation of RECQL4 SP sites were analyzed by Western blotting. d CDK1/2 inhibitors repress p-SP level of RECQL4. HEK293T cells expressing 3×FLAG-RECQL4 were pretreated with 10 µM RO3306 (CDK1i), 10 µM CDK2i-III (CDK2i) or combination of CDK1i and CDK2i for 4 h. Then, 3×FLAG-tagged RECQL4 were purified and SP sites’ phosphorylation was analyzed by Western blotting. e Ser89/Ser251 phosphorylation promotes recruitment of RECQL4 to laser-induced DSBs. GFP-tagged RECQL4 Wt, RQ4-2A, and RQ4-2D were expressed in U2OS cells, and the cells were stripped with micro-point laser after treatment with CDK1 and CDK2 inhibitors or DMSO. The relative intensities of these cells are presented as mean ± s.e.m. The number of cells were quantified, for RQ4Wt, n = 9; RQ4Wt with CDK1/2i, n = 16; RQ4-2A, n = 19; RQ4-2A with CDK1/2i, n = 17; RQ4-2D, n = 26. RQ4-2D with CDK1/2i, n = 19. Scale bar, 5 µm. f Inhibition of CDK1 and CDK2 attenuated recruitment of YFP-MRE11 to laser-induced DSBs. For DMSO, n = 27; for CDK1/2i, n = 49. g Inhibition of CDK1 and CDK2 represses RECQL4’s interaction with MRE11. The U2OS cells pretreated with CDK1 and CDK2 inhibitors or DMSO were irradiated for 10 Gy and processed for IP with anti-MRE11 antibody 5 min after irradiation. h Ser89/Ser251 phosphorylation promotes the interaction of RECQL4 with MRE11. GFP-tagged RECQL4, RQ4-2A, and RQ4-2D were immunoprecipitated from HEK293T cells after a 10 Gy IR treatment. MRE11 and Ku70 were measured in the IP products