Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear i nterior is limited to passage through the nuclear pores. Here we demonstrat e the nuclear uptake of extended linear DNA molecules by a combination of f luorescence microscopy and single-molecule manipulation techniques, using t he latter to follow uptake kinetics of individual molecules in real time. T he assays were carried out on nuclei reconstituted in vitro from extracts o f Xenopus eggs, which provide both a complete complement of biochemical fac tors involved in nuclear protein import, and unobstructed access to the nuc lear pores. We find that uptake of DNA is independent of ATP or GTP hydroly sis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic: considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction releva nt to the uptake process.