DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.     

Image analysis-based measurement of DNA supercoiling changes in transformed and nontransformed human cell lines

An image analysis system was used to visualize and measure the changes in nucleoid diameter (nuclear matrix core plus extruded DNA loops) which occur when increasing concentrations of propidium iodide are used to titrate the DNA supercoiling response. Parallel core size measurements allow estimates of the changes in apparent DNA loop size. Unlike sedimentation assays, DNA loop size estimates are not influenced by particle mass, require no prior cell labeling, and can be performed on a per cell basis. This technique was used to examine changes in DNA loop characteristics which may occur when cells are transformed or undergo changes in their proliferative state. SV40-transformation of human diploid fibroblast lines resulted in a significant increase in both the nucleoid core and average DNA loop size. Lymphoblast cell lines also had larger nucleoid dimensions than normal lymphocytes. The response of several established human tumor cell lines indicated slightly increased loop but not core sizes as compared to normal human diploid fibroblasts. Changes in proliferative state also resulted in changes in DNA loop characteristics as measured in this assay. Both quiescent fibroblasts and unstimulated lymphocytes appeared to have smaller or more condensed DNA loop structures than their proliferating counterparts. These results demonstrate the utility of this assay in detecting changes in DNA loop structure which occur in association with changes in the proliferative activity of cells in culture.     

Differential immunoreactivity for Z-DNA in rat myoblast nuclei during their terminal differentiation

L6 myoblasts in vitro accomplish the process of terminal differentiation from dividing mononucleated cells to quiescent plurinucleated myotubes, synthesizing muscle-specific proteins. They have been tested, using paraformaldehyde and acetic acid fixations and immunocytochemical techniques, for the presence of Z-DNA at different stages: namely after 3, 5 and 8-9 days of culture. The nuclei of the actively dividing 3-day myoblasts were strongly Z-DNA and B-DNA-positive. The inhibition of replication by araC did not diminish the reaction. In the myotubes, the nuclei became Z-DNA-negative but were still B-DNA-positive. In contrast, the nuclei of a non-fusing alpha-amanitin-resistant mutant (Ama102) stayed Z-DNA-positive. These results tend to show that during the process of terminal differentiation Z-DNA either becomes less accessible or is present in undetectable amounts. In circular DNAs, it has been shown that the presence of Z-DNA depends on their negative supercoiling. In addition, the presence of closed superhelical loops of nuclear DNA has been demonstrated in several mammalian cell types; moreover, the density of DNA topological turns in these loops varies during cellular differentiation and malignant transformation. The relationship between these results and ours is discussed.