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 interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of f...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2001-06, Vol.98 (13), p.7247-7252
Main Authors: Salman, H., Zbaida, D., Rabin, Y., Chatenay, D., Elbaum, M.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Animals
Bacteriophages
Biochemistry
Biological Sciences
Biology
Cell nucleus
Cell Nucleus - metabolism
Cell Nucleus - ultrastructure
Cells
Deoxyribonucleic acid
DNA
DNA - metabolism
Eggs
Female
GTP
Guanosine Triphosphate - metabolism
Humans
In Vitro Techniques
Kinetics
Microscopy, Fluorescence
Models, Theoretical
Molecules
Nuclear membrane
Nuclear pore
Nuclear Pore - metabolism
Nuclear Pore - ultrastructure
Oocytes - cytology
Oocytes - metabolism
Physics
Protein Transport
Serum Albumin - metabolism
Serum Albumin, Bovine - metabolism
Tweezers
Wheat Germ Agglutinins - pharmacology
Xenopus laevis
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Summary:Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, 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 relevant to the uptake process.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.121067698