Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52.
When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies.
Di Primio
Cristina
C
Laboratorio di Terapia Genica e Molecolare, Istituto di Fisiologia Clinica Area della Ricerca del CNR, via Moruzzi 1, 56124 Pisa, Italy.
Galli
Alvaro
A
Cervelli
Tiziana
T
Zoppè
Monica
M
Rainaldi
Giuseppe
G
D002467:Cell Nucleus / Q000737:chemistry; D002999:Clone Cells; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins