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Use of RecA fusion proteins to induce genomic modifications in zebrafish.

Liao HK, Essner JJ - Nucleic Acids Res. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
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.

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A model for gene targeting using complementary ss DNA-NLS-RecA-Gal4. Both sense and antisense single-strand (ss-s and ss-a)-NLS-RecA-Gal4 filaments are co-injected into zebrafish embryos. The RecA homology search activity can guide the filaments to the targeted region. The cssDNA-NLS-RecA-Gal4 filaments can undergo homologous pairing and strand invasion. This causes the formation of D-loops that are stabilized by the Gal4 dimerization domains between the complementary filaments on the target chromosome. This compact DNA joint molecule is assumed to block replication fork progression, leading to a DNA DSB.
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Figure 6: A model for gene targeting using complementary ss DNA-NLS-RecA-Gal4. Both sense and antisense single-strand (ss-s and ss-a)-NLS-RecA-Gal4 filaments are co-injected into zebrafish embryos. The RecA homology search activity can guide the filaments to the targeted region. The cssDNA-NLS-RecA-Gal4 filaments can undergo homologous pairing and strand invasion. This causes the formation of D-loops that are stabilized by the Gal4 dimerization domains between the complementary filaments on the target chromosome. This compact DNA joint molecule is assumed to block replication fork progression, leading to a DNA DSB.

Mentions: We have demonstrated that complementary ssDNA-NLS-RecA-Gal4 filaments targeted to the gol locus are able to induce LOH at this locus after injection into zebrafish embryos. We show that the NLS-RecA-Gal4 protein is able to coat cssDNA (Supplementary Figure S1). When cssDNA–protein complex is injected into the zebrafish embryos, the cssDNA-NLS-RecA-Gal4 filaments likely undergo a homology search to find homologous chromosomal DNA, similar to the behavior of native RecA. In support of this, we did not detect LOH at the gol locus when cssDNA-NLS-RecA-Gal4 filaments complementary to vegfa, floating head and prom1a were used (Table 1), suggesting that the targeted disruption of the gol locus was dependent upon the DNA sequence in the css-gol DNA-NLS-RecA-Gal4 filaments. We propose a model for this activity where the css-gol DNA-NLS-RecA-Gal4 filaments target the gol locus for disruption by creating arrested replication forks that result in DSBs (Figure 6). Alternatively, the cssDNA-NLS-RecA-Gal4 filaments could promote recruitment of components of the DNA repair pathway and endogenous nucleases to specific chromosomal sites, resulting in deletions or site-specific insertion of exogenous DNA.Figure 6.


Use of RecA fusion proteins to induce genomic modifications in zebrafish.

Liao HK, Essner JJ - Nucleic Acids Res. (2011)

A model for gene targeting using complementary ss DNA-NLS-RecA-Gal4. Both sense and antisense single-strand (ss-s and ss-a)-NLS-RecA-Gal4 filaments are co-injected into zebrafish embryos. The RecA homology search activity can guide the filaments to the targeted region. The cssDNA-NLS-RecA-Gal4 filaments can undergo homologous pairing and strand invasion. This causes the formation of D-loops that are stabilized by the Gal4 dimerization domains between the complementary filaments on the target chromosome. This compact DNA joint molecule is assumed to block replication fork progression, leading to a DNA DSB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105420&req=5

Figure 6: A model for gene targeting using complementary ss DNA-NLS-RecA-Gal4. Both sense and antisense single-strand (ss-s and ss-a)-NLS-RecA-Gal4 filaments are co-injected into zebrafish embryos. The RecA homology search activity can guide the filaments to the targeted region. The cssDNA-NLS-RecA-Gal4 filaments can undergo homologous pairing and strand invasion. This causes the formation of D-loops that are stabilized by the Gal4 dimerization domains between the complementary filaments on the target chromosome. This compact DNA joint molecule is assumed to block replication fork progression, leading to a DNA DSB.
Mentions: We have demonstrated that complementary ssDNA-NLS-RecA-Gal4 filaments targeted to the gol locus are able to induce LOH at this locus after injection into zebrafish embryos. We show that the NLS-RecA-Gal4 protein is able to coat cssDNA (Supplementary Figure S1). When cssDNA–protein complex is injected into the zebrafish embryos, the cssDNA-NLS-RecA-Gal4 filaments likely undergo a homology search to find homologous chromosomal DNA, similar to the behavior of native RecA. In support of this, we did not detect LOH at the gol locus when cssDNA-NLS-RecA-Gal4 filaments complementary to vegfa, floating head and prom1a were used (Table 1), suggesting that the targeted disruption of the gol locus was dependent upon the DNA sequence in the css-gol DNA-NLS-RecA-Gal4 filaments. We propose a model for this activity where the css-gol DNA-NLS-RecA-Gal4 filaments target the gol locus for disruption by creating arrested replication forks that result in DSBs (Figure 6). Alternatively, the cssDNA-NLS-RecA-Gal4 filaments could promote recruitment of components of the DNA repair pathway and endogenous nucleases to specific chromosomal sites, resulting in deletions or site-specific insertion of exogenous DNA.Figure 6.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Iowa State University, Ames, IA 50011, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus