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HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Abstract

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.

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Figure 1: HCLK2 interacts with components of the S-phase checkpoint.
Figure 2: HCLK2-deficient cells exhibit S-phase checkpoint defects.
Figure 3: HCLK2 is required for damage-induced FANCD2 monoubiquitination and recruitment to sites of replication stress.
Figure 4: C. elegans clk-2 mutants and HCLK2-deficient human cells exhibit similar defects in FANCD2 function and ICL repair.
Figure 5: HCLK2 is required for homologous-recombination repair following replication stress.
Figure 6: HCLK2 depletion compromises claspin phosphorylation and Cdc25A degradation in the absence of HCLK2, but not Chk2 pThr 68 phosphorylation.
Figure 7: Chk1 instability in HCLK2-depleted cells is dependent on ATR phosphorylation of Chk1 at Ser 345.
Figure 8: Schematic representation of the role of HCLK2 in regulating the S-phase checkpoint.

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Acknowledgements

We wish to thank: A. Jones and H. Cooper for mass-spectrometry; Y. Murakawa and S. Takeda for laser micro-irradiation experiments; S. Hekimi, S. West, N. Lakin, G. Smith, S. Jackson and A. Jazayeri for HCLK2, Rad51, ATR, ATRIP and Cdc25A antibodies, respectively; N. Mailand, J. Bartek, H. Piwnica-Worms, Y.-W. Zhang and B. Abraham for Chk1 constructs; H. Bryant and T. Helleday for SW480sn3 cells, ISce-1 and GFP-reporter plasmids; N. O'Reilly for peptide synthesis; and R. Peat and R. Horton-Harpin for cell culture. Thanks to J. Svejstrup, P.Zegerman, A. Jazayeri, G. Alderton, J. Falck, P. Robins, M. Segurado and members of the Boulton lab for technical advice and comments on the manuscript. This work was funded by Breast Cancer Campaign (GA3221) and Cancer Research UK.

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S.J.C. performed the majority of experiments. L.J.B., A.J.C., J.S.M. and J.D.W. all contributed to the experiments. S.J.B., S.J.C. and L.J.B. contributed intellectually to this work.

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Correspondence to Simon J. Boulton.

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The authors declare no competing financial interests.

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Collis, S., Barber, L., Clark, A. et al. HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability. Nat Cell Biol 9, 391–401 (2007). https://doi.org/10.1038/ncb1555

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