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

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation pa st the enzyme. During this reaction, the enzyme remains bound to the recogn ition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visu alised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction in termediates with DNA topoisomerases. A DNA cleavage-deficient mutant of Eco AI, which has normal DNA translocation and ATPase activities, was used in t hese DNA supercoiling assays. Ln the presence of eubacterial DNA topoisomer ase I, which specifically removes negative supercoils, the EcoAI mutant int roduced positive supercoils into relaxed plasmid DNA substrate in a reactio n dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positiv e supercoils were not seen when eubacterial DNA topoisomerase I was replace d by eukaryotic DNA topoisomerase I, which removes both positive and negati ve supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to t he product of the supercoiling reaction resulted in its rapid relaxation. T hese results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates po sitive supercoils ahead and negative supercoils behind the moving complex i n the contracting and expanding DNA loops, respectively. In addition, we sh ow that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed D NA. This suggests that the topological changes in the DNA substrate associa ted with DNA translocation by type I restriction enzymes do not appear to b e the trigger for DNA cleavage. (C) 2000 Academic Press.