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Use of RecA fusion proteins to induce genomic modifications in zebrafish
The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA fila...
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2011
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105420/ https://www.ncbi.nlm.nih.gov/pubmed/21266475 http://dx.doi.org/10.1093/nar/gkq1363 |
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author | Liao, Hsin-Kai Essner, Jeffrey J. |
author_facet | Liao, Hsin-Kai Essner, Jeffrey J. |
author_sort | Liao, Hsin-Kai |
collection | PubMed |
description | The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB. |
format | Text |
id | pubmed-3105420 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-31054202011-06-01 Use of RecA fusion proteins to induce genomic modifications in zebrafish Liao, Hsin-Kai Essner, Jeffrey J. Nucleic Acids Res Genome Integrity, Repair and Replication The bacterial recombinase RecA forms a nucleic acid-protein filament on single-stranded (ss) DNA during the repair of double-strand breaks (DSBs) that efficiently undergoes a homology search and engages in pairing with the complementary DNA sequence. We utilized the pairing activity of RecA–DNA filaments to tether biochemical activities to specific chromosomal sites. Different filaments with chimeric RecA proteins were tested for the ability to induce loss of heterozygosity at the golden locus in zebrafish after injection at the one-cell stage. A fusion protein between RecA containing a nuclear localization signal (NLS) and the DNA-binding domain of Gal4 (NLS-RecA-Gal4) displayed the most activity. Our results demonstrate that complementary ssDNA filaments as short as 60 nucleotides coated with NLS-RecA-Gal4 protein are able to cause loss of heterozygosity in ∼3% of the injected embryos. We demonstrate that lesions in ∼9% of the F0 zebrafish are transmitted to subsequent generations as large chromosomal deletions. Co-injection of linear DNA with the NLS-RecA-Gal4 DNA filaments promotes the insertion of the DNA into targeted genomic locations. Our data support a model whereby NLS-RecA-Gal4 DNA filaments bind to complementary target sites on chromatin and stall DNA replication forks, resulting in a DNA DSB. Oxford University Press 2011-05 2011-01-25 /pmc/articles/PMC3105420/ /pubmed/21266475 http://dx.doi.org/10.1093/nar/gkq1363 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), 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 Liao, Hsin-Kai Essner, Jeffrey J. Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title | Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title_full | Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title_fullStr | Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title_full_unstemmed | Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title_short | Use of RecA fusion proteins to induce genomic modifications in zebrafish |
title_sort | use of reca fusion proteins to induce genomic modifications in zebrafish |
topic | Genome Integrity, Repair and Replication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105420/ https://www.ncbi.nlm.nih.gov/pubmed/21266475 http://dx.doi.org/10.1093/nar/gkq1363 |
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