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

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 protei...

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Autores principales: Deshpande, Rajashree A., Marin-Gonzalez, Alberto, Barnes, Hannah K., Woolley, Phillip R., Ha, Taekjip, Paull, Tanya T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10505227/
https://www.ncbi.nlm.nih.gov/pubmed/37717054
http://dx.doi.org/10.1038/s41467-023-41544-8
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author Deshpande, Rajashree A.
Marin-Gonzalez, Alberto
Barnes, Hannah K.
Woolley, Phillip R.
Ha, Taekjip
Paull, Tanya T.
author_facet Deshpande, Rajashree A.
Marin-Gonzalez, Alberto
Barnes, Hannah K.
Woolley, Phillip R.
Ha, Taekjip
Paull, Tanya T.
author_sort Deshpande, Rajashree A.
collection PubMed
description 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.
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spelling pubmed-105052272023-09-18 Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice Deshpande, Rajashree A. Marin-Gonzalez, Alberto Barnes, Hannah K. Woolley, Phillip R. Ha, Taekjip Paull, Tanya T. Nat Commun Article 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. Nature Publishing Group UK 2023-09-16 /pmc/articles/PMC10505227/ /pubmed/37717054 http://dx.doi.org/10.1038/s41467-023-41544-8 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Deshpande, Rajashree A.
Marin-Gonzalez, Alberto
Barnes, Hannah K.
Woolley, Phillip R.
Ha, Taekjip
Paull, Tanya T.
Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title_full Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title_fullStr Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title_full_unstemmed Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title_short Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
title_sort genome-wide analysis of dna-pk-bound mrn cleavage products supports a sequential model of dsb repair pathway choice
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10505227/
https://www.ncbi.nlm.nih.gov/pubmed/37717054
http://dx.doi.org/10.1038/s41467-023-41544-8
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