Istituto di Fisiologia Clinica     
Kalvala A., Rainaldi G., Di Primio C., Liverani V., Falaschi A., Galli A. Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein. In: Nucleic Acids Research, vol. Aug;38 (14) article n. e149.. Oxford University Press, 2010.
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 overexpression 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 nonhomologous 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 toincrease GT in human cells and a 'protein delivery approach' offers a new tool for developing novel strategies for genome modification and gene therapy applications.
Subject Homologous recombination, yeast, gene targeting, Protein delivery

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