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Targeted ubiquitination of CDT1 by the DDB1–CUL4A–ROC1 ligase in response to DNA damage

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Abstract

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates1,2,3,4. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1–CUL1, the DDB1–CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4ADDB1, in response to UV irradiation.

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Figure 1: DDB1 binds to an N-terminal region in CUL4A.
Figure 2: DDB1 and CAND1 bind to CUL4A in a mutually exclusive manner.
Figure 3: DDB1 binds to CDT1 and bridges CDT1 to CUL4A.
Figure 4: DDB1 targets CDT1 for CUL4A-mediated ubiquitination.

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Acknowledgements

We thank P. Raychaudhuri for providing the DDB1 expression vector; X. Wu for providing a CDT1 expression vector and a GST–CDT1 antibody; N. Zheng for providing purified DDB1 protein; S. Jackson for generating the CUL4A antibody; and M. Furukawa and members of the Xiong laboratory for the discussion and help throughout the course of this study. C.M.M. is a recipient of the George H. Hitchings New Investigator Award in Health Research and Training of the Triangle Community Foundation. This work is supported by National Institutes of Health grant GM067113 to Y.X.

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  1. Tomohiko Ohta

    Present address: Division of Breast and Endocrine Surgery, St Marianna University School of Medicine, Kawasaki, 216-8511, Japan

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, 27599-7295, NC, USA

    Jian Hu, Chad M. McCall, Tomohiko Ohta & Yue Xiong

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  1. Jian Hu
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Correspondence to Yue Xiong.

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Hu, J., McCall, C., Ohta, T. et al. Targeted ubiquitination of CDT1 by the DDB1–CUL4A–ROC1 ligase in response to DNA damage. Nat Cell Biol 6, 1003–1009 (2004). https://doi.org/10.1038/ncb1172

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