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Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

Author

Listed:
  • Rajashree A. Deshpande

    (Encoded Therapeutics)

  • Alberto Marin-Gonzalez

    (Johns Hopkins University School of Medicine
    Howard Hughes Medical Institute)

  • Hannah K. Barnes

    (The University of Texas at Austin)

  • Phillip R. Woolley

    (The University of Texas at Austin)

  • Taekjip Ha

    (Johns Hopkins University School of Medicine
    Howard Hughes Medical Institute)

  • Tanya T. Paull

    (The University of Texas at Austin)

Abstract

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

Suggested Citation

  • Rajashree A. Deshpande & Alberto Marin-Gonzalez & Hannah K. Barnes & Phillip R. Woolley & Taekjip Ha & Tanya T. Paull, 2023. "Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41544-8
    DOI: 10.1038/s41467-023-41544-8
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    References listed on IDEAS

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    1. Zhongxuan Zhang & Xiaoxiao Rong & Tianjin Xie & Zehao Li & Haozhi Song & Shujun Zhen & Haifeng Wang & Jiahui Wu & Samie R. Jaffrey & Xing Li, 2024. "Fluorogenic CRISPR for genomic DNA imaging," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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