Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo

Abstract

Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene approximately 3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast.

Figure 1

Altered RAD51 genes and analyses of gene or protein expression. (A) RAD51 mutant plasmids. Plasmid pYNRAD51(K342E) contains a mutation in RAD51 cDNA that will result in the substitution of K(aag, Lys) with E(gag, Glu) at amino acid position 342. This substitution increases its interaction with Rad54; the I345T substitution increases the DNA binding activity of Rad51; the Y388H substitution and the G393D substitution each decrease the interaction of Rad51 with Rad52. pYN132 is an empty vector as indicated in the Materials and Methods. (B) The MMS sensitivity assay. Yeast cells carrying the indicated plasmids were cultured and serial dilutions were made on YPD plates containing 0 or 0.025% of MMS to assess survival.

Figure 1

Altered RAD51 genes and analyses of gene or protein expression. (A) RAD51 mutant plasmids. Plasmid pYNRAD51(K342E) contains a mutation in RAD51 cDNA that will result in the substitution of K(aag, Lys) with E(gag, Glu) at amino acid position 342. This substitution increases its interaction with Rad54; the I345T substitution increases the DNA binding activity of Rad51; the Y388H substitution and the G393D substitution each decrease the interaction of Rad51 with Rad52. pYN132 is an empty vector as indicated in the Materials and Methods. (B) The MMS sensitivity assay. Yeast cells carrying the indicated plasmids were cultured and serial dilutions were made on YPD plates containing 0 or 0.025% of MMS to assess survival.

Figure 2

The expression of RAD51 allele from various pYNRAD51 plasmids. (A) RT–PCR analysis of total RNA extracts from LSY678(Int) strain harboring various pYNRAD51 mutant plasmids. A 1.2 kb RAD51 product was amplified using primers of Rad51F and Rad51B and electrophoresed through 1% agarose gel. (B) Western blot analysis of whole-cell extracts from LSY402(Δrad51) and LSY678(Int) containing the various pYNRAD51 expression plasmids. Protein extracts from LSY402(Δrad51) or LSY678(Int), alone or containing the different pYNRAD51 expression plasmids, were evaluated by blotting with rabbit α Rad51 antibody and visualized by an anti-rabbit antibody conjugated with HRP in an enhanced chemiluminescence ECL™ system. Protein extracts from LSY402(Δrad51) with empty vector (pYN132) were used as a negative control. An internal control was set up by utilizing a goat β-actin antibody and visualized by anti-goat IgG-HRP antibody in the same ECL™ system.

Figure 2

The expression of RAD51 allele from various pYNRAD51 plasmids. (A) RT–PCR analysis of total RNA extracts from LSY678(Int) strain harboring various pYNRAD51 mutant plasmids. A 1.2 kb RAD51 product was amplified using primers of Rad51F and Rad51B and electrophoresed through 1% agarose gel. (B) Western blot analysis of whole-cell extracts from LSY402(Δrad51) and LSY678(Int) containing the various pYNRAD51 expression plasmids. Protein extracts from LSY402(Δrad51) or LSY678(Int), alone or containing the different pYNRAD51 expression plasmids, were evaluated by blotting with rabbit α Rad51 antibody and visualized by an anti-rabbit antibody conjugated with HRP in an enhanced chemiluminescence ECL™ system. Protein extracts from LSY402(Δrad51) with empty vector (pYN132) were used as a negative control. An internal control was set up by utilizing a goat β-actin antibody and visualized by anti-goat IgG-HRP antibody in the same ECL™ system.

Figure 3

(A) Gene targeting system for chromosomal nucleotide repair. Plasmid pAUR101(Int)Hyg^s-eGFP, which carries a Hyg^s-eGFP mutant cassette, was integrated into LSY678. The wild-type hygromycin gene contains TAT at codon 46, while the mutant gene contains a TAG, and the correction creates a TAC codon, restoring hygromycin resistance and eGFP expression. The single-stranded DNA oligonucleotide vector Hyg3S/74NT—74 nt long and containing three phosphorothioate linkages at each end—is designed to target the non-transcribed strand of the fusion gene, repairing codon 46. The targeting nucleotide in oligonucleotide Hyg3S/74NT was highlighted. (B) Evidence of eGFP expression from the Hyg-eGFP fusion gene in LSY678(Int) bearing pYNRAD51(K342E). Yeast cells observed by Zeiss LSM510 confocal microscope (1) before and (2) after the cells were targeted with Hyg3S/74NT. (C) DNA sequence confirmation of fusion gene correction. Direct sequence from PCR amplification showed that the TAG mutation in the integrated Hyg^s-eGFP cassette was corrected. Wild-type (TAT), mutant (TAG) and corrected (TAC) sequences are provided. The corrected colony sequence comes from an experiment in which pYNRAD51(K342E) was overexpressed in LSY678(Int).

Figure 3

(A) Gene targeting system for chromosomal nucleotide repair. Plasmid pAUR101(Int)Hyg^s-eGFP, which carries a Hyg^s-eGFP mutant cassette, was integrated into LSY678. The wild-type hygromycin gene contains TAT at codon 46, while the mutant gene contains a TAG, and the correction creates a TAC codon, restoring hygromycin resistance and eGFP expression. The single-stranded DNA oligonucleotide vector Hyg3S/74NT—74 nt long and containing three phosphorothioate linkages at each end—is designed to target the non-transcribed strand of the fusion gene, repairing codon 46. The targeting nucleotide in oligonucleotide Hyg3S/74NT was highlighted. (B) Evidence of eGFP expression from the Hyg-eGFP fusion gene in LSY678(Int) bearing pYNRAD51(K342E). Yeast cells observed by Zeiss LSM510 confocal microscope (1) before and (2) after the cells were targeted with Hyg3S/74NT. (C) DNA sequence confirmation of fusion gene correction. Direct sequence from PCR amplification showed that the TAG mutation in the integrated Hyg^s-eGFP cassette was corrected. Wild-type (TAT), mutant (TAG) and corrected (TAC) sequences are provided. The corrected colony sequence comes from an experiment in which pYNRAD51(K342E) was overexpressed in LSY678(Int).

Figure 3

(A) Gene targeting system for chromosomal nucleotide repair. Plasmid pAUR101(Int)Hyg^s-eGFP, which carries a Hyg^s-eGFP mutant cassette, was integrated into LSY678. The wild-type hygromycin gene contains TAT at codon 46, while the mutant gene contains a TAG, and the correction creates a TAC codon, restoring hygromycin resistance and eGFP expression. The single-stranded DNA oligonucleotide vector Hyg3S/74NT—74 nt long and containing three phosphorothioate linkages at each end—is designed to target the non-transcribed strand of the fusion gene, repairing codon 46. The targeting nucleotide in oligonucleotide Hyg3S/74NT was highlighted. (B) Evidence of eGFP expression from the Hyg-eGFP fusion gene in LSY678(Int) bearing pYNRAD51(K342E). Yeast cells observed by Zeiss LSM510 confocal microscope (1) before and (2) after the cells were targeted with Hyg3S/74NT. (C) DNA sequence confirmation of fusion gene correction. Direct sequence from PCR amplification showed that the TAG mutation in the integrated Hyg^s-eGFP cassette was corrected. Wild-type (TAT), mutant (TAG) and corrected (TAC) sequences are provided. The corrected colony sequence comes from an experiment in which pYNRAD51(K342E) was overexpressed in LSY678(Int).