Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination

MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus MC, DR Rotterdam, The Netherlands.
The EMBO Journal (Impact Factor: 10.43). 10/2004; 23(17):3548-58. DOI: 10.1038/sj.emboj.7600353
Source: PubMed


Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

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    • "A small fraction of p53 +/− and p53 −/− embryos also display a defect in neural tube closure, which later (around E13.5) develops as exencephaly (Sah et al., 1995). Homozygous deletion of checkpoint control genes Hus1 and Rad17 in mouse embryos leads to neural tube defects at E9.5 and E11.5, respectively (Weiss et al., 2000; Budzowska et al., 2004). Failure of neural tube closure might be a byproduct of a general disturbance in development, but the fact that Mrad9b is expressed in the embryonic brain at E8.5–9.5 supports the hypothesis that Mrad9b is specifically involved in brain formation. "
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    ABSTRACT: RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b(-/-) embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b(+/-) embryos exhibit abnormal neural tube closure. Mrad9b(-/-) mouse embryonic fibroblasts are not viable. Mrad9b(-/-) ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b(+/+) controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b(-/-) cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents.
    Full-text · Article · Nov 2010 · Developmental Dynamics
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    • "For the targeting efficiency assay, ES cells were cultured in BRL-conditioned medium as described elsewhere [46]. WT and the Rad21+/− ES cell lines were transfected with a Rad54-GFP knockin construct [20]. One week after selection with Puromycin, single-cell suspensions of surviving colonies were made following trypsinization and analysis by FACS on a green fluorescence (eGFP) versus forward scatter (FSC-H) plot. "
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    ABSTRACT: Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were identified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/- animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/- animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues.
    Full-text · Article · Aug 2010 · PLoS ONE
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    • "Yeast genetic analyses implicated the importance of Rad17 in cell-cycle arrest and cellular survival in response to DNA damage and DNA replication blockage (Paulovich et al, 1997). Rad17-knockout mouse displayed embryonic lethality with numerous developmental defects (Budzowska et al, 2004). Targeted deletion of Rad17 in HCT116 and DT40 cells inhibits mitotic as well as S-phase damage checkpoint functions and further results in chromosomal aberration and endoreduplication (Wang et al, 2003; Kobayashi et al, 2004). "
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    ABSTRACT: Recent studies have shown a critical function for the ubiquitin-proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non-biased assay to identify fast-turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells. Our characterization showed that UV-induced Rad17 oscillation is mediated by Cdh1/APC, a ubiquitin-protein ligase. Studies using a degradation-resistant Rad17 mutant demonstrated that Rad17 stabilization prevents the termination of checkpoint signalling, which in turn attenuates the cellular re-entry into cell-cycle progression. The findings provide an insight into how the proteolysis of Rad17 by Cdh1/APC regulates the termination of checkpoint signalling and the recovery from genotoxic stress.
    Full-text · Article · May 2010 · The EMBO Journal
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