Mechanism of inhibition of DNA synthesis by cycloheximide in [formula omitted] cells

Abstract

The mechanisms underlying DNA chain elongation were investigated in Balb 3 T3 cells utilizing both intact cells and a whole cell lysate system. Incorporation of labeled deoxynucleoside triphosphates into short (<10 S) chains greatly exceeded that into long (>10 S) chains up to 1 min of incubation with lysate suggesting that chain growth occurs discontinuously along both strands of DNA. Both the kinetics of incorporation and pulse-chase data showed that short chains were true precursors to large molecular weight DNA. Alkaline sucrose sedimentation analysis of DNA synthesized by the lysate system revealed that Okazaki fragments, 5 S in size, matured into 8 S fragments prior to the formation of large molecular weight DNA. Exposure of cells to 25 μM cycloheximide for 1 h reduced protein and DNA synthesis to 5% and 25%, respectively, of control. Prior inhibition of protein synthesis by cycloheximide delayed the maturation of 5 S fragments into 8 S intermediates but did not significantly affect the joining of 8 S pieces to form large molecular weight DNA in vitro. Alkaline sucrose sedimentation profiles of DNA from cycloheximide-treated cells pulse-labeled with [ 3H]thymidine were significantly different from those of control cells indicating altered initiation and termination patterns of DNA replication in the presence of the inhibitor. On the basis of these data and other evidence in literature, it is proposed that a combination of reduced rate of replicon initiation and impaired maturation of 5 S fragments into 8 S intermediates accounts for the observed decreases in the overall rate of DNA synthesis and in the rate of chain elongation in cycloheximide-treated cells.