PROKARYOTIC DNA LIGASES UNWIND SUPERHELICAL DNA

We have studied the effect on DNA topology of binding of prokaryotic D NA ligases (T4 and E. coil) to superhelical or nicked circular DNA. Pe rforming topoisomerase I-mediated relaxation in the presence of increa sing amounts of T4 ligase led to a shift in the topoisomer distributio n to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of ni cked circular molecules by E. coil DNA ligase in the presence of incre asing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Pe rforming a similar experiment with reverse partners, using E. coil DNA ligase as ligand, and T4 ligase as sealing agent, we observed that th e E. coil enzyme also unwound the DNA. Thus, prokaryotic DNA ligases c an be added to an ever-growing list of DNA-binding proteins that unwin d the DNA upon binding. (C) 1996 Academic Press, Inc.