Chain Elongation of DNA and Joining of DNA Intermediates in Intact and Permeabilized Mouse Cells

Abstract

When the sequence of DNA chain elongation in intact mouse cells was analyzed by pulselabeling the cells with [³H]thymidine followed by sedimentation in an alkaline sucrose gradient, four classes of DNA, different in size, were observed. They were the Okazaki-type initial fragment, two high molecular weight DNA classes which we designated as DNA intermediates I and II, and the bulk of chromosomal DNA. When the size of DNA synthesized in permeabilized cells (cells treated with detergent to make them permeable to nucleoside triphosphates) was analyzed by the same method, we found that one of the intermediate DNAs, DNA intermediate I, was the major product of the in vitro DNA replication and further elongation of the DNA chain from DNA intermediate I to II was lacking. But when a soluble fraction released from the cells after treatment with Triton X-100 was added to the permeabilized cells, the activity of joining chains of DNA intermediate I to form DNA intermediate II was partially restored. From the size of the two DNA intermediates and the way they were elongated as revealed by the sedimentation analysis, DNA intermediate I and II seem to correspond to replicon-size DNA and clustered replicon-size DNA, respectively. And our results suggest that there exists some unknown factor or process which is required for the joining of completed replicon-size DNA at the terminals.