Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein

Abstract

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a 'protein delivery approach' offers a new tool for developing novel strategies for genome modification and gene therapy applications.

Figure 1.

Purification, ssDNA binding activity and delivery of yRad52tat11. (A) Maps of the yRad52tat11 wild-type fusion protein, the Y66A and the R70A mutant, that for simplicity only show the N-terminal DNA binding domain; the numbers represent amino acid residues in the wild-type yeast molecule; the ssDNA binding domain is shown. Both mutations are located in this domain; the tat11 and the his-tag are located at the C-terminus. (B) Coomassie staining of purified wild-type-yRad52-tat11 protein (about 52 kDa); M-lane, markers; BSA-lanes 1 and 2, 200 and 400 ng of bovine serum albumin; I-lane, 5 µg of total protein extract from IPTG-induced bacteria; NI-lane, 5 µg of total protein extract from not induced bacteria; WT-lanes 1, 2 and 3, wild-type yRad52tat11 purified protein elutions; the yield was 80, 70 and 50 ng/μl for three different elutions. (C) In vitro mobility shift assay with ssDNA (30 µM as nucleotides) and increasing concentrations (0.05, 0.1, 0.2, 0.3 µM) of wt yRad52tat11 protein or GFPtat11 (0.3 µM) as control; the reaction mixtures were loaded in a 0.9% agarose gel, electropheresed and stained with ethidium bromide. (D) In vitro mobility shift assay with ssDNA (30 µM as nucleotides) alone, in presence of 0.3 µM concentration of wild-type yRad52 or GFPtat11. (E) In vitro mobility shift assay with ssDNA (30 µM as nucleotides) alone or in presence of 0.3 µM concentration of wild-type yRad52tat11, R70A or Y66A mutated protein, as indicated. (F) Western blot analysis of total cell extracts; 6 × 10⁵ HeLaG1 cells were incubated with 20 µg/ml wild-type-yRad52tat11 or mutant proteins for 24 h; cell lysis and total cell extracts were carried out as reported in ‘Materials and Methods’ section; 10 µg of total proteins were loaded in each well; the blot was hybridized with anti-penta-6-histidine antibody; lane 1, extract from wild-type yRad52tat11-exposed cells; lane 2, extract from R70A treated cells; lane 3, extract from Y66A treated cells; tubulin was used as loading control. (G) Western blot analysis of nuclear extracts from 3 × 10⁶ total cells treated with 20 µg/ml of wild type or mutated yRad52tat11 protein for 24 h; aliquots corresponding to 10 µg of nuclear protein extract were loaded and electrophoresed; lane 1, 1 µg of purified yRad52tat11protein; lane 2, nuclear extract from Y66A mutant-treated cells; lane3, nuclear extract from wild-type yRad52tat11-treated cells; lane 4, nuclear extract from R70A mutant-treated cells.

Figure 2.

Effect of yRad52tat11 on intrachromosomal recombination. (A) HeLaG1 cells contain two copies of HygR genes inactivated by 10-bp insertions, either at a unique PvuI site (hyg1) or at a unique SacII site (hyg2); the two mutated hyg genes are in direct repeat orientation and are separated by a sequence containing the amino-glycoside phosphotransferase (Neo) gene conferring resistance to G418; an intrachromosomal recombination event occurring by gene conversion between the two hyg sequences results in restoration of one of the mutant hyg genes to wild type; the intrachromosomal deletion of the DNA sequence between the two mutated hyg genes leads to the formation of a HygR wild type (Hyg WT) with loss of intervening sequence; the intrachromosomal recombination was measured after incubating HeLaG1 cells with 12, 20 or 30 µg/ml yRad52tat11 proteins for 24 h (B) or 48 h (C). The frequency of intrachromosomal recombination was determined as total HygR clones per 10⁵ viable cells; the data are reported as mean of three to five independent experiments ± standard deviation; GFPtat11 (12, 20 and 30 µg/ml) was used as negative control (C in B and C); WT, wild-type protein; M1 and M2, Y66A and R70A mutant proteins, respectively.

Figure 3.

Effect of yRad52tat11 protein on GT and RI. (A) Measure of GT events between the single chromosomal HygR gene utilized in the experiments of Figure 2 (hyg1), and the BamHI fragment transfected by electroporation to the cells, corresponding to mutant hyg2 in the same figure; a GT event leads to the formation of wild-type HygR gene (Hyg WT) with the restoration of the restriction site PvuI. (B) GT experiment in HeLa1B cells carried out transfecting by electroporation 10 μg of BamHI hyg2 DNA fragment purified from agarose gel; cells were pre-incubated for 6 h with 16, 24 or 30 μg/ml yRad52tat11 or Y66A protein (M); GT frequency is expressed as total HygR clones per 10⁵ viable cells and the results are the mean of at least three independent experiments ± standard deviation. (C) The 359 bp Hyg fragment was amplified by PCR from the genomic DNA of HygR clones and digested with PvuI; the digestion gave a 289- and a 70-bp fragment; lanes 1–6, DNA from HygR clones digested with PvuI; lane 7, not restricted DNA. (D) Effect of wild-type yRad52tat11 or Y66A protein (M) on random integration; the frequency of random integration was determined in HeLa1B cells by electroporating 8 µg of plasmid DNA and measured as number of total Bla^R clones per 10⁴ viable cells; the results are the mean of four independent experiments ± standard deviation.