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Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has be...

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Autores principales: Gonçalves, Manuel A. F. V., van Nierop, Gijsbert P., Holkers, Maarten, de Vries, Antoine A. F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333848/
https://www.ncbi.nlm.nih.gov/pubmed/22189101
http://dx.doi.org/10.1093/nar/gkr1234
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author Gonçalves, Manuel A. F. V.
van Nierop, Gijsbert P.
Holkers, Maarten
de Vries, Antoine A. F.
author_facet Gonçalves, Manuel A. F. V.
van Nierop, Gijsbert P.
Holkers, Maarten
de Vries, Antoine A. F.
author_sort Gonçalves, Manuel A. F. V.
collection PubMed
description The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has been primarily on the former type of DNA lesions mainly due to the paucity of SSB-based recombination models. Here, to investigate the role of nicked DNA molecules as HR-initiating substrates in human somatic cells, we devised a homology-directed gene targeting system based on exogenous donor and chromosomal target DNA containing recognition sequences for the adeno-associated virus sequence- and strand-specific endonucleases Rep78 and Rep68. We found that HR is greatly fostered if a SSB is not only introduced in the chromosomal acceptor but also in the donor DNA template. Our data are consistent with HR models postulating the occurrence of SSBs or single-stranded gaps in both donor and acceptor molecules during the genetic exchange process. These findings can guide the development of improved HR-based genome editing strategies in which sequence- and strand-specific endonucleolytic cleavage of the chromosomal target site is combined with that of the targeting vector.
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spelling pubmed-33338482012-04-23 Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells Gonçalves, Manuel A. F. V. van Nierop, Gijsbert P. Holkers, Maarten de Vries, Antoine A. F. Nucleic Acids Res Genome Integrity, Repair and Replication The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has been primarily on the former type of DNA lesions mainly due to the paucity of SSB-based recombination models. Here, to investigate the role of nicked DNA molecules as HR-initiating substrates in human somatic cells, we devised a homology-directed gene targeting system based on exogenous donor and chromosomal target DNA containing recognition sequences for the adeno-associated virus sequence- and strand-specific endonucleases Rep78 and Rep68. We found that HR is greatly fostered if a SSB is not only introduced in the chromosomal acceptor but also in the donor DNA template. Our data are consistent with HR models postulating the occurrence of SSBs or single-stranded gaps in both donor and acceptor molecules during the genetic exchange process. These findings can guide the development of improved HR-based genome editing strategies in which sequence- and strand-specific endonucleolytic cleavage of the chromosomal target site is combined with that of the targeting vector. Oxford University Press 2012-04 2011-12-20 /pmc/articles/PMC3333848/ /pubmed/22189101 http://dx.doi.org/10.1093/nar/gkr1234 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Genome Integrity, Repair and Replication
Gonçalves, Manuel A. F. V.
van Nierop, Gijsbert P.
Holkers, Maarten
de Vries, Antoine A. F.
Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title_full Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title_fullStr Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title_full_unstemmed Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title_short Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells
title_sort concerted nicking of donor and chromosomal acceptor dna promotes homology-directed gene targeting in human cells
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333848/
https://www.ncbi.nlm.nih.gov/pubmed/22189101
http://dx.doi.org/10.1093/nar/gkr1234
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