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Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

Author

Listed:
  • Mariia Mikhova

    (Michigan State University
    Michigan State University)

  • Noah J. Goff

    (Michigan State University
    Michigan State University)

  • Tomáš Janovič

    (Michigan State University)

  • Joshua R. Heyza

    (Michigan State University)

  • Katheryn Meek

    (Michigan State University
    Michigan State University)

  • Jens C. Schmidt

    (Michigan State University
    Michigan State University)

Abstract

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115 Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop an approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and allowed us to estimate the overall repair capacity of the NHEJ pathway in living cells.

Suggested Citation

  • Mariia Mikhova & Noah J. Goff & Tomáš Janovič & Joshua R. Heyza & Katheryn Meek & Jens C. Schmidt, 2024. "Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-54545-y
    DOI: 10.1038/s41467-024-54545-y
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    References listed on IDEAS

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    1. Gabrielle J. Grundy & Stuart L. Rulten & Raquel Arribas-Bosacoma & Kathryn Davidson & Zuzanna Kozik & Antony W. Oliver & Laurence H. Pearl & Keith W. Caldecott, 2016. "The Ku-binding motif is a conserved module for recruitment and stimulation of non-homologous end-joining proteins," Nature Communications, Nature, vol. 7(1), pages 1-11, September.
    2. Ineke Brouwer & Gerrit Sitters & Andrea Candelli & Stephanie J. Heerema & Iddo Heller & Abinadabe J. Melo de & Hongshan Zhang & Davide Normanno & Mauro Modesti & Erwin J. G. Peterman & Gijs J. L. Wuit, 2016. "Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA," Nature, Nature, vol. 535(7613), pages 566-569, July.
    3. Siyu Chen & Linda Lee & Tasmin Naila & Susan Fishbain & Annie Wang & Alan E. Tomkinson & Susan P. Lees-Miller & Yuan He, 2021. "Structural basis of long-range to short-range synaptic transition in NHEJ," Nature, Nature, vol. 593(7858), pages 294-298, May.
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