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Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains...

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Autores principales: Whelan, Donna R., Rothenberg, Eli
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980414/
https://www.ncbi.nlm.nih.gov/pubmed/33707212
http://dx.doi.org/10.1073/pnas.2021963118
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author Whelan, Donna R.
Rothenberg, Eli
author_facet Whelan, Donna R.
Rothenberg, Eli
author_sort Whelan, Donna R.
collection PubMed
description Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.
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spelling pubmed-79804142021-03-26 Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination Whelan, Donna R. Rothenberg, Eli Proc Natl Acad Sci U S A Biological Sciences Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair. National Academy of Sciences 2021-03-16 2021-03-11 /pmc/articles/PMC7980414/ /pubmed/33707212 http://dx.doi.org/10.1073/pnas.2021963118 Text en Copyright © 2021 the Author(s). Published by PNAS. http://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biological Sciences
Whelan, Donna R.
Rothenberg, Eli
Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title_full Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title_fullStr Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title_full_unstemmed Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title_short Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
title_sort super-resolution mapping of cellular double-strand break resection complexes during homologous recombination
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980414/
https://www.ncbi.nlm.nih.gov/pubmed/33707212
http://dx.doi.org/10.1073/pnas.2021963118
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