Mediator of DNA Damage Checkpoint Protein 1 Regulates BRCA1 Localization and Phosphorylation in DNA Damage Checkpoint Control
ABSTRACT 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.
- SourceAvailable from: Pierre Caron
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- "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. "
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.Nucleic Acids Research 12/2013; 42(3). DOI:10.1093/nar/gkt1309 · 9.11 Impact Factor
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- "siRNA transfection was performed as described previously . 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. "
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.PLoS ONE 05/2012; 7(5):e36520. DOI:10.1371/journal.pone.0036520 · 3.23 Impact Factor
DNA Repair and Human Health, 10/2011; , ISBN: 978-953-307-612-6
- "DSBs usually result from insults such as X-or gamma rays or topoisomerase poisons, or simply arise when a replication fork encounters damaged DNA (Hartlerode & Scully, 2009). DSB repair takes place in vivo within defined foci characterized by a distinctive histone phosphorylation (γ‐H2AX), accumulation of autophosphorylated DNA‐PKcs and recruitment of repair and signaling proteins, including 53BP1, NFBD1/MDC1 and the chromatin‐bound form of the MRE11/RAD50/NBS1 complex (Chan et al., 2002a; Lou et al., 2003; Mirzoeva & Petrini, 2001; Paull et al., 2000; Schultz et al., 2000; Shang et al., 2003). Two mechanisms have evolved to remediate this type of damage: non-homologous end joining (NHEJ) and homologous recombination (HR) (Hartlerode & Scully, 2009). "