Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.
Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.
Ziemienowicz
A
A
Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland.
Görlich
D
D
Lanka
E
E
Hohn
B
B
Rossi
L
L
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007473:Ion Channels; D008322:Mammals; D014162:Transfection / Q000379:methods*; D037521:Virulence Factors
D001426:Bacterial Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007473:Ion Channels; D037521:Virulence Factors; C051583:virE2 protein, Agrobacterium