Nuclear reconstitution in vitro: stages of assembly around protein-free DNA |
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| Authors: | J Newport |
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| Institution: | 1. The National Institute of Health and Family Welfare (NIHFW), New Delhi 110067, India;2. Jamia Millia Islamia (JMI), New Delhi 110025, India;1. School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India;2. School of Life Sciences, Jawaharlal Nehru University, New Delhi, India;1. Center for Molecular Prevention and Environmental Medicine, Department of Preventive Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan;2. Central Institute for Experimental Animals, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, Japan;3. Department of Neurology, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan;4. Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Shimokasuya, Isehara, Kanagawa, Japan;3. Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada;5. Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada;4. Centres for Research in Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada;6. Research in Mass Spectrometry, York University, Toronto, Ontario M3J 1P3, Canada |
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| Abstract: | We have developed a cell-free system derived from Xenopus eggs that reconstitutes nuclear structure around an added protein-free substrate (bacteriophage lambda DNA). Assembled nuclei are morphologically indistinguishable from normal eukaryotic nuclei: they are surrounded by a double membrane containing nuclear pores and are lined with a peripheral nuclear lamina. Nuclear assembly involves discrete intermediate steps, including nucleosome assembly, scaffold assembly, and nuclear membrane and lamina assembly, indicating that during reconstitution nuclear organization is assembled one level at a time. Topoisomerase II inhibitors block nuclear assembly. Lamin proteins and membrane vesicles bind to chromatin late in assembly, suggesting that these components do not interact with chromatin that is formed early in assembly. Reconstituted nuclei replicate their DNA; replication begins only after envelope formation has initiated, indicating that envelope attachment may be important for regulating replication. |
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