Book of Abstracts: Albany 2003
June 17-21 2003
Nuclear Uptake of DNA
Macromolecular communication between cytoplasm and nucleus in eukaryotic cells takes place via the nuclear pore complexes. Water, ions, nucleotides, and other small chemical species pass the nuclear pore by diffusion, while most proteins and RNAs are ferried through the pore by specific transport receptors. Beyond the normal physiological transport substrates, many virus infecting animal cells require nuclear import of their genomic DNA or RNA. Similarly, most strategies for medical gene therapy depend on nuclear uptake of exogenous DNA, often to non-dividing cells via the nuclear pores. These considerations motivate studies of DNA uptake by fluorescence microscopy and single-molecule manipulation using optical tweezers (1). The latter permit measurement of the uptake kinetics of individual molecules. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus laevis eggs. This system provides at once a complete cellular biochemistry and unobstructed access to the nuclear pores. We find that DNA import to the nucleus is independent of ATP or GTP hydrolysis, but like protein import it can be blocked by wheat germ agglutinin. The uptake kinetics are much slower than would be expected from simple hydrodynamic considerations. A simple model suggests that a large friction in the pore dominates the uptake process, which is driven by femto-Newton forces.
Department of Materials and Interfaces