Mediator of DNA Damage Checkpoint Protein 1 Regulates BRCA1 Localization and Phosphorylation in DNA Damage Checkpoint Control

Department of Oncology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2003; 278(16):13599-602. DOI: 10.1074/jbc.C300060200
Source: PubMed


BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.

Full-text preview

Available from:
  • Source
    • "All other siRNAs were purchased from Eurofins MWG Operon. siRNA sequences are as follows: siControl: AAUUCUCCGAACGUGUCACGUdTdT (26); siCtIP: GCUAAAACAGGAACGAAUCdTdT (4); siMre11: ACAGGAGAAGAGAUCAACUdTdT (26); siSOSS-A:CGUGAUGGCAUGAAUAUUGdTdT (27); siExo1: UAGUGUUUCAGGAUCAACAUCAUCUdTdT (28); siDNA-PKcs: CUUUAUGGUGGCCAUGGAGdTdT (29); siBRCA1: GGAACCUGUCTCCACAAAGdTdT (30); si53BP1: GGACUCCAGUGUUGUCAUUdTdT (31). The efficiency of gene knockdown was examined by western blotting and DSB resection was measured 48 h after transfection. "
    [Show abstract] [Hide abstract]
    ABSTRACT: 5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.
    Full-text · Article · Dec 2013 · Nucleic Acids Research
  • Source
    • "Cells were transiently depleted of endogenous PALB2, or BRCA1 or RAP80, using siRNAs against the 39-UTR or the coding sequence, respectively, as described (Ganesan et al., 2002;Hu et al., 2011;Zhang et al., 2009a). For other depletion experiments, siRNAs (59–39) directed against FANCJ (GUACAGUACCCCACCUUAU) (Zhang et al., 2010), CtIP (GCUAA- AACAGGAACGAAUC) (Yu and Chen, 2004), Abraxas (GUAAAAGGU- GAAGCCAAGA) (Liu et al., 2007), RNF8 (GGACAAUUAUGGACAACAA) (Mailand et al., 2007), MDC1 (UCCAGUGAAUCCUUGAGGU) (Lou et al., 2003), and NBS1 (GGAGGAAGAUGUCAAUGUU) (Yoo et al., 2009) were utilized as previously described. All siRNAs were purchased from Dharmacon. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The PALB2 protein is associated with breast cancer susceptibility and Fanconi anemia. Notably, PALB2 is also required for DNA repair by homologous recombination (HR). The mechanisms that regulate PALB2, and the functional significance of its interaction with the BRCA1 breast cancer susceptibility protein, are poorly understood, however. Here, to better understand these processes, we fused PALB2, or the PALB2(L21P) mutant which cannot bind to BRCA1, with the BRCT repeats that are present in, and which localize, BRCA1. Our results yield important insight into the regulation of PALB2 function. Both fusion proteins can bypass BRCA1 to localize to sites of DNA damage. Further, the localized fusion proteins are functional, as determined by their ability to support the assembly of RAD51 foci, even in the absence of the capacity of PALB2 to bind BRCA1. Strikingly, the localized fusion proteins mediate DNA double-strand break (DSB)-initiated HR and resistance to mitomycin C in PALB2-deficient cells. Additionally, we show that the BRCA1-PALB2 heterodimer, rather than the PALB2-PALB2 homodimer, mediates these responses. Importantly, we offer the first insight into how BRCA1-dependent recruitment of PALB2 is integrated with other DNA damage signaling pathways. We find that PALB2 localization depends on the presence of MDC1, RNF8, RAP80, and Abraxas upstream of BRCA1. Thus, PALB2 may link HR to a key ubiquitin-related signaling pathway that responds to DSBs.
    Full-text · Article · Oct 2012 · Journal of Cell Science
  • Source
    • "siRNA transfection was performed as described previously [20]. Briefly, cells were grown in six-well plate to 60% confluence and immediately before transfection washed with serum free medium, and 800 µl of serum-free medium were added per well. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.
    Full-text · Article · May 2012 · PLoS ONE
Show more