High Molecular Weight DNA Intermediates Synthesized by Permeabilized L Cells

Mouse L cells rendered permeable to deoxynucleoside triphosphates synthesize DNA as an extension of replication forks that were active in the intact cells. The permeabilization process does not affect the size of the bulk cell DNA. Intermediate molecular weight DNA, synthesized in the intact cells, is neither degraded to small molecular weight DNA nor processed into bulk cell DNA following the permeabilization process. DNA synthesized by the permeable cells demonstrates a heterogenous distribution in alkaline sucrose gradients, with peaks at 26 S and 71 S. Pulse-chase experiments demonstrate that these two classes of DNA intermediates do not have a precursor product relation. They appear to be synthesized independently and at the same rate. The results are compatible with continuous synthesis of DNA along one template strand and discontinuous synthesis along the opposite strand. DNA synthesis in isolated L cell nuclei was compared to the process in permeabilized cells with the results demonstrating that the rate of DNA synthesis is slower in the nuclei than it is in the permeable cells. Alkaline sucrose gradient studies demonstrate that the DNA synthesized by isolated nuclei is smaller than the DNA synthesized by the permeable cells.