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

Rajashree A. Deshpande, Alberto Marin-Gonzalez, Hannah K. Barnes, Phillip R. Woolley, Taekjip Ha and Tanya T. Paull ()
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Rajashree A. Deshpande: Encoded Therapeutics
Alberto Marin-Gonzalez: Johns Hopkins University School of Medicine
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
Tanya T. Paull: The University of Texas at Austin

Nature Communications, 2023, vol. 14, issue 1, 1-17

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-41544-8

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