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Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Resection of the 5′-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradat...

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Autores principales: Bazzano, Dominic, Lomonaco, Stephanie, Wilson, Thomas E
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8682756/
https://www.ncbi.nlm.nih.gov/pubmed/34263309
http://dx.doi.org/10.1093/nar/gkab597
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author Bazzano, Dominic
Lomonaco, Stephanie
Wilson, Thomas E
author_facet Bazzano, Dominic
Lomonaco, Stephanie
Wilson, Thomas E
author_sort Bazzano, Dominic
collection PubMed
description Resection of the 5′-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5′ ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60–70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.
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spelling pubmed-86827562021-12-20 Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo Bazzano, Dominic Lomonaco, Stephanie Wilson, Thomas E Nucleic Acids Res NAR Breakthrough Article Resection of the 5′-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5′ ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60–70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases. Oxford University Press 2021-07-15 /pmc/articles/PMC8682756/ /pubmed/34263309 http://dx.doi.org/10.1093/nar/gkab597 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle NAR Breakthrough Article
Bazzano, Dominic
Lomonaco, Stephanie
Wilson, Thomas E
Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title_full Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title_fullStr Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title_full_unstemmed Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title_short Mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
title_sort mapping yeast mitotic 5′ resection at base resolution reveals the sequence and positional dependence of nucleases in vivo
topic NAR Breakthrough Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8682756/
https://www.ncbi.nlm.nih.gov/pubmed/34263309
http://dx.doi.org/10.1093/nar/gkab597
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