Abstract
ParE is the ATP-binding subunit of topoisomerase IV (Topo IV). During topoisomerization, 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 hydrolyze 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 IV proteins exhibited an elevated level of spontaneous DNA cleavage that could be associated with a decreased rate of DNA resealing. In ParE T201A Topo IV, this defect appeared to result from an increased likelihood that the tetrameric enzyme would fall apart after DNA cleavage. Thus, while ATP is not required for DNA cleavage, the properties of these mutant enzymes suggests that ATP-hydrolysis informs DNA religation.
Highlights
ParE is the ATP-binding subunit of topoisomerase IV (Topo IV)
As a result of a screen for dominant-negative mutations of parE [5], we have isolated and characterized three mutant ParE proteins defective in either ATP binding or ATP hydrolysis when reconstituted with ParC to form Escherichia coli topoisomerase IV (Topo IV)
Require ATP—In the first article in this series [5], we described the isolation of dominant-negative parE mutants and the initial characterization of the superhelical DNA relaxation activity of Topo IVs reconstituted with the mutant ParEs and ParC
Summary
ParE is the ATP-binding subunit of topoisomerase IV (Topo IV). During topoisomerization, the ATP-binding and hydrolysis cycle must be coordinated with the cycle of DNA cleavage and religation. Type II topoisomerases couple the energy of ATP hydrolysis to alter the linking number of DNA [1,2,3] To do so, these enzymes must execute an ordered sequence of conformational changes and chemical reactions: trapping a segment of DNA, passage of the trapped segment through a transient doublestrand break in another DNA segment, release of the passed DNA segment from the interior of the enzyme, and resetting of the enzyme for another round of catalysis. In the case of ParE T201A Topo IV, which can bind but not hydrolyze ATP, hyper-DNA cleavage can be correlated with a decrease in the rate of resealing of the cleaved DNA and an increased chance that the tetrameric enzyme will fall apart after cleavage, leaving ParC linked covalently to the DNA This suggests that, under normal circumstances, ATP hydrolysis is required for the DNA gate to close
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