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bioRxiv · 10.1101/2022.12.07.519501

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

Deshpande, R. A.·Marin-Gonzalez, A.·Ha, T.·Paull, T. T.
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Abstract

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' 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 the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency 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 much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both 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.

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Deshpande, R. A., Marin-Gonzalez, A., Ha, T., Paull, T. T.. 2022-12-07. Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice. https://doi.org/10.1101/2022.12.07.519501

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