Mutational analysis of Escherichia coli topoisomerase IV. ATPase negative mutants of ParE induce hyper-DNA cleavage

ParE is the ATP-binding subunit of topoisomerase TV (Topo TV). During topoi somerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation. We have isolated three dominant- negative mutant alleles of parE that encode ParE proteins that fail to hydr olyze ATP when reconstituted with ParC to form Topo IV. ParE G110S Topo IV and ParE S123L Topo IV failed to bind ATP at all, whereas ParE T201A could bind ATP. All three mutant Topo TV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing. In ParE T201A Topo TV, this defect appeared to result from a n increased likelihood that the tetrameric enzyme would fall apart after DN A cleavage. Thus, while ATP is not required for DNA cleavage, the propertie s of these mutant enzymes suggests that ATP-hydrolysis informs DNA religati on.