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Disruption of the Rad9/Rad1/Hus1 (9–1–1) complex leads to checkpoint signaling and replication defects

Oncogene volume 23, pages 5586–5593 (2004)Cite this article

Abstract

The checkpoint sliding-clamp complex, Rad9/Rad1/Hus1, plays a critical role during initiation of checkpoint signals in response to DNA damage and replication disruption. We investigated the impact of loss of Rad1 on checkpoint function and on DNA replication in mammalian cells. We show that RAD1 is an essential gene for sustained cell proliferation and that loss of Rad1 causes destabilization of Rad9 and Hus1 and consequently disintegration of the sliding-clamp complex. In Rad1-depleted cells, Atr-dependent Chk1 activation was impaired whereas Atm-mediated Chk2 activation was unaffected, suggesting that the sliding clamp is required primarily in Atr-dependent signal activation. Disruption of sliding-clamp function also caused a major defect in S-phase control. Rad1-depleted cells exhibited an RDS phenotype, indicating that damage-induced S-phase arrest was compromised by Rad1 loss. Furthermore, lack of Rad1 also affected the efficiency of replication recovery from DNA synthesis blockage, resulting in a prolonged S phase. These deficiencies may perpetually generate DNA strand breakage as we have found chromosomal abnormalities in Rad1-depleted cells. We conclude that the Rad9/Rad1/Hus1 complex is essential for Atr-dependent checkpoint signaling, which may play critical roles in the facilitation of DNA replication and in the maintenance of genomic integrity.

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Acknowledgements

We thank Howard Lieberman (Columbia University) for antibodies to Rad9 and Hus1 and Robert Weiss (Cornell University) for the Hus1−/−/p21−/− MEF cells. David R Engelke (University of Michigan) kindly provided the pTZU6+1 vector containing a U6 promotor. We also thank Lee Zou (Harvard Medical School) for critical reading of this manuscript and helpful discussions. This work was supported by Public Health Service grants from the National Cancer Institute (CA76172 and CA91029 to LL).

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Authors and Affiliations

  1. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, 77030, TX, USA

    Shilai Bao, Xin Wang, Huyong Zheng & Lei Li

  2. Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, 77030, TX, USA

    Tao Lu & Walter N Hittelman

  3. Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, 77030, TX, USA

    Li-E Wang & Qingyi Wei

  4. Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, Houston, 77030, TX, USA

    Lei Li

  5. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Anwai Datun Road, Beijing, 100101, China

    Shilai Bao

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Correspondence to Lei Li.

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Bao, S., Lu, T., Wang, X. et al. Disruption of the Rad9/Rad1/Hus1 (9–1–1) complex leads to checkpoint signaling and replication defects. Oncogene 23, 5586–5593 (2004). https://doi.org/10.1038/sj.onc.1207753

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