Abstract
Mouse L Cells, grown in suspension culture can be rendered permeable to exogenous deoxynucleoside triphosphates by a cold shock in a near isotonic buffer system. These cells use the deoxynucleotides to synthesize DNA in a semiconservative fashion. The addition of 0.05% Triton X-100 to this system increases the permeability of the cells so that exogenously supplied macromolecules gain access to the DNA. When DNAase and phosphodiesterase are added to the detergent-permeabilized cells, the cell DNA is rapidly degraded, demonstrating that the enzymes reach the DNA within the first 2 min of the incubation period. Addition of whole calf thymus histone or histone fractions to the detergent-permeabilized cells inhibits DNA synthesis. The lysine-rich histone, F 1 is a more effective inhibitor than the arginine-rich histone, F 3. The other histone fractions including the slightly lysine-rich fractions, F 2a and F 2b, are intermediate between F 1 and F 3 as inhibitors of DNA synthesis. Kinetic analysis demonstrates that the added histones increase apparent K m and reduce V of DNA synthesis in the permeabilized cells. These studies suggest the possibility that histones alter the association of the DNA replication complex and the DNA template in a manner that reduces the rate of DNA synthesis.
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