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Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

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  • معلومة اضافية
    • المصدر:
      Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE
    • بيانات النشر:
      Original Publication: London : Nature Publishing Group, copyright 2011-
    • الموضوع:
      DNA Breaks, Double-Stranded* ; Ovum* ; Tissue Extracts* ; Xenopus*; DNA-Binding Proteins/*chemistry ; DNA-Binding Proteins/*physiology ; Signal Transduction/*genetics ; Signal Transduction/*physiology; Animals ; Ataxia Telangiectasia Mutated Proteins/genetics ; Ataxia Telangiectasia Mutated Proteins/metabolism ; Cell Cycle/genetics ; Cell Cycle/physiology ; Checkpoint Kinase 1/metabolism ; DNA/genetics ; DNA/metabolism ; Phosphorylation/genetics
    • نبذة مختصرة :
      The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1's nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.
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    • Grant Information:
      R01 GM122887 United States GM NIGMS NIH HHS
    • الرقم المعرف:
      0 (DNA-Binding Proteins)
      0 (Tissue Extracts)
      9007-49-2 (DNA)
      EC 2.7.1.- (Atr protein, Xenopus)
      EC 2.7.11.1 (Ataxia Telangiectasia Mutated Proteins)
      EC 2.7.11.1 (Checkpoint Kinase 1)
    • الموضوع:
      Date Created: 20210112 Date Completed: 20210826 Latest Revision: 20210826
    • الموضوع:
      20250114
    • الرقم المعرف:
      PMC7801695
    • الرقم المعرف:
      10.1038/s41598-020-80626-1
    • الرقم المعرف:
      33432091