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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) RecA fusion proteins used in this study. (B) Injection of complementary ssDNA-NLS-RecA-Gal4 filaments leads to LOH at the gol locus, resulting in mosaic eye pigmentation. Genotype of the embryos used for injection is shown. (C) An example of LOH at the gol locus. Dorsal views of eyes at 3 dpf showing wild-type pigmentation patterns in a non-injected embryo (upper panel) and mosaic pigmentation patterns (bottom panel) after injection of complementary ssgol-NLS-RecA-Gal4 targeting filaments. (D) DNA complementary to either exons4/5 or exon6 of gol was used for making gol RecA filaments. Golm1381 (m, mutant; 1381, 1381 bp) and golex6m-60 (ex6, exon 6; m, mutant; 60, 60 bp) DNAs carry mutations resulting in a stop codon (asterisk). Golex6-60-1(ex6, exon 6; 60, 60 bp) and golex6-60-2 (ex6, exon 6; 60, 60 bp) oligonucleotides do not have mutations.
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Figure 1: (A) RecA fusion proteins used in this study. (B) Injection of complementary ssDNA-NLS-RecA-Gal4 filaments leads to LOH at the gol locus, resulting in mosaic eye pigmentation. Genotype of the embryos used for injection is shown. (C) An example of LOH at the gol locus. Dorsal views of eyes at 3 dpf showing wild-type pigmentation patterns in a non-injected embryo (upper panel) and mosaic pigmentation patterns (bottom panel) after injection of complementary ssgol-NLS-RecA-Gal4 targeting filaments. (D) DNA complementary to either exons4/5 or exon6 of gol was used for making gol RecA filaments. Golm1381 (m, mutant; 1381, 1381 bp) and golex6m-60 (ex6, exon 6; m, mutant; 60, 60 bp) DNAs carry mutations resulting in a stop codon (asterisk). Golex6-60-1(ex6, exon 6; 60, 60 bp) and golex6-60-2 (ex6, exon 6; 60, 60 bp) oligonucleotides do not have mutations.

Mentions: Native RecA was purchased from Sigma. Different RecA fusion proteins were cloned for this study (Figure 1A). For the different fusion proteins, the full-length sequence encoding bacterial RecA and the yeast Gal4 DNA-binding domain were amplified with the proofreading polymerase Pfx50 (Invitrogen) from pBEU14 RecA (13) and pGBT9 Gal4 plasmids (Clontech), respectively. For all proteins produced, the N-terminus of RecA was fused with a NLS from SV40 Large T Antigen to promote nuclear targeting (14). The NLS at the 5′-terminus of each fusion protein was added using the following primer: 5′-CATATGCCACCTAAAAAGAAGAGAAAGGTAGAAGACCCCAAGATGGCTATCGACGAAAACAA-3′. NLS-RecA was amplified using the 5′-primer and the following 3′-primer: 5′-GATCGCGGCCGCAAAATCTTCGTTAGTTTCTG-3′. The primers used to prepare the 3′-terminal of different forms of RecA fused to Gal4 were 5′-GCGGCCGCCGATACAGTCAACTGTCTTT-3′ for NLSRecAGal4 and 5′-CAGCGGAGACCTTTTGGTTTTG-3′ for NLS-RecA-Gal4ΔDD. The NLS-RecA sequence was fused to the Gal4 DNA-binding domain sequence via a unique NheI restriction enzyme site using the following oligonucleotides: 5′-GCTAGCAAAATCTTCGTTAGTTTCTG-3′ and 5′-GCTAGCATGAAGCTACTGTCTTCTATCG-3′. The purified polymerase chain reaction (PCR) products were ligated to NdeI and NotI restriction sites in the pET41a expression vector (Novagen). This vector contains a sequence coding for an 8xHis-affinity tag after the NotI site at the C-terminus.Figure 1.


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

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

(A) RecA fusion proteins used in this study. (B) Injection of complementary ssDNA-NLS-RecA-Gal4 filaments leads to LOH at the gol locus, resulting in mosaic eye pigmentation. Genotype of the embryos used for injection is shown. (C) An example of LOH at the gol locus. Dorsal views of eyes at 3 dpf showing wild-type pigmentation patterns in a non-injected embryo (upper panel) and mosaic pigmentation patterns (bottom panel) after injection of complementary ssgol-NLS-RecA-Gal4 targeting filaments. (D) DNA complementary to either exons4/5 or exon6 of gol was used for making gol RecA filaments. Golm1381 (m, mutant; 1381, 1381 bp) and golex6m-60 (ex6, exon 6; m, mutant; 60, 60 bp) DNAs carry mutations resulting in a stop codon (asterisk). Golex6-60-1(ex6, exon 6; 60, 60 bp) and golex6-60-2 (ex6, exon 6; 60, 60 bp) oligonucleotides do not have mutations.
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Related In: Results  -  Collection

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Figure 1: (A) RecA fusion proteins used in this study. (B) Injection of complementary ssDNA-NLS-RecA-Gal4 filaments leads to LOH at the gol locus, resulting in mosaic eye pigmentation. Genotype of the embryos used for injection is shown. (C) An example of LOH at the gol locus. Dorsal views of eyes at 3 dpf showing wild-type pigmentation patterns in a non-injected embryo (upper panel) and mosaic pigmentation patterns (bottom panel) after injection of complementary ssgol-NLS-RecA-Gal4 targeting filaments. (D) DNA complementary to either exons4/5 or exon6 of gol was used for making gol RecA filaments. Golm1381 (m, mutant; 1381, 1381 bp) and golex6m-60 (ex6, exon 6; m, mutant; 60, 60 bp) DNAs carry mutations resulting in a stop codon (asterisk). Golex6-60-1(ex6, exon 6; 60, 60 bp) and golex6-60-2 (ex6, exon 6; 60, 60 bp) oligonucleotides do not have mutations.
Mentions: Native RecA was purchased from Sigma. Different RecA fusion proteins were cloned for this study (Figure 1A). For the different fusion proteins, the full-length sequence encoding bacterial RecA and the yeast Gal4 DNA-binding domain were amplified with the proofreading polymerase Pfx50 (Invitrogen) from pBEU14 RecA (13) and pGBT9 Gal4 plasmids (Clontech), respectively. For all proteins produced, the N-terminus of RecA was fused with a NLS from SV40 Large T Antigen to promote nuclear targeting (14). The NLS at the 5′-terminus of each fusion protein was added using the following primer: 5′-CATATGCCACCTAAAAAGAAGAGAAAGGTAGAAGACCCCAAGATGGCTATCGACGAAAACAA-3′. NLS-RecA was amplified using the 5′-primer and the following 3′-primer: 5′-GATCGCGGCCGCAAAATCTTCGTTAGTTTCTG-3′. The primers used to prepare the 3′-terminal of different forms of RecA fused to Gal4 were 5′-GCGGCCGCCGATACAGTCAACTGTCTTT-3′ for NLSRecAGal4 and 5′-CAGCGGAGACCTTTTGGTTTTG-3′ for NLS-RecA-Gal4ΔDD. The NLS-RecA sequence was fused to the Gal4 DNA-binding domain sequence via a unique NheI restriction enzyme site using the following oligonucleotides: 5′-GCTAGCAAAATCTTCGTTAGTTTCTG-3′ and 5′-GCTAGCATGAAGCTACTGTCTTCTATCG-3′. The purified polymerase chain reaction (PCR) products were ligated to NdeI and NotI restriction sites in the pET41a expression vector (Novagen). This vector contains a sequence coding for an 8xHis-affinity tag after the NotI site at the C-terminus.Figure 1.

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