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C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains

Author

Listed:
  • Ragini Bhargava

    (Beckman Research Institute of the City of Hope
    Beckman Research Institute of the City of Hope)

  • Manbir Sandhu

    (Beckman Research Institute of the City of Hope
    Beckman Research Institute of the City of Hope)

  • Sanychen Muk

    (Beckman Research Institute of the City of Hope)

  • Gabriella Lee

    (Beckman Research Institute of the City of Hope)

  • Nagarajan Vaidehi

    (Beckman Research Institute of the City of Hope
    Beckman Research Institute of the City of Hope)

  • Jeremy M. Stark

    (Beckman Research Institute of the City of Hope
    Beckman Research Institute of the City of Hope)

Abstract

To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1–2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.

Suggested Citation

  • Ragini Bhargava & Manbir Sandhu & Sanychen Muk & Gabriella Lee & Nagarajan Vaidehi & Jeremy M. Stark, 2018. "C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains," Nature Communications, Nature, vol. 9(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-04867-5
    DOI: 10.1038/s41467-018-04867-5
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    Cited by:

    1. Sandra Wimberger & Nina Akrap & Mike Firth & Johan Brengdahl & Susanna Engberg & Marie K. Schwinn & Michael R. Slater & Anders Lundin & Pei-Pei Hsieh & Songyuan Li & Silvia Cerboni & Jonathan Sumner &, 2023. "Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    2. Bert van de Kooij & Alex Kruswick & Haico van Attikum & Michael B. Yaffe, 2022. "Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    3. Zhiqian Li & Lang You & Anita Hermann & Ethan Bier, 2024. "Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier," Nature Communications, Nature, vol. 15(1), pages 1-19, December.
    4. Martin Peterka & Nina Akrap & Songyuan Li & Sandra Wimberger & Pei-Pei Hsieh & Dmitrii Degtev & Burcu Bestas & Jack Barr & Stijn Plassche & Patricia Mendoza-Garcia & Saša Šviković & Grzegorz Sienski &, 2022. "Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
    5. Metztli Cisneros-Aguirre & Felicia Wednesday Lopezcolorado & Linda Jillianne Tsai & Ragini Bhargava & Jeremy M. Stark, 2022. "The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened," Nature Communications, Nature, vol. 13(1), pages 1-17, December.

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