Mutational analysis of Escherichia coli topoisomerase IV. Selection of dominant-negative parE alleles

In order to define regions of ParE, one of the two subunits of topoisomeras e IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles. Both wild -type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid, Mutated parE alleles were rescued fro m those plasmids that caused IPTG-dependent cell death, The mutant ParE pro teins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that af fected covalent complex formation.