Chapter 21 Nucleic Acid Synthesis in Permeabilized Eukaryotic Cells

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Permeable cell systems are developed in bacteria and disrupted or permeable cell systems are developed in eukaryotes to allow exogenously supplied deoxynucleoside triphosphates and other reaction components to be supplied directly to the replication complex functioning on its intrinsic DNA template. The technique described for rendering eukaryotic cells permeable to nucleotides has several advantages in the study of nucleic acid synthesis under near physiological conditions. The technique itself is simple; the cells remain in a monodisperse suspension and are easy to pipet quantitatively. The cells are freely permeable to phosphorylated compounds which gain rapid access to the nucleus. Nucleotides are incorporated into DNA without intermediate breakdown and rephosphorylation. DNA synthesis in permeable cells is semiconservative, the products are high-molecularweight DNA intermediates, and DNA synthesis occurs as extensions of replication sites that were active in vivo. This technique provides an assay system in which the polymerases function on their endogenous templates. It should be useful in determining whether agents that affect nucleic acid synthesis in intact cells exert their effects by direct action on the polymerase or on the template. The system should also be useful for studies of the intermediates in nucleic acid synthesis, and finally for studies of physiological changes in the activities of polymerases that occur with metabolic manipulation of the cells. As the addition of Triton X-100 renders the cells permeable to exogenous proteins, this technique may also be useful in complementation studies of eukaryotic cells with genetic defects in DNA synthesis.