Mutational Analysis of Escherichia coliTopoisomerase IV

Abstract

In order to define regions of ParE, one of the two subunits of topoisomerase 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 from those plasmids that caused IPTG-dependent cell death. The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation.Type II topoisomerases utilize the cycle of ATP binding, hydrolysis of the ␤-␥ phosphodiester bond, and release of ADP and P i to drive a series of conformational changes that allow the enzyme to pass one DNA helix through a transient proteinbridged, double-strand break in either another segment of the same DNA helix or a different DNA helix. This results in an alteration of the linking number of the DNA. In general, if the same DNA ring contains both the segment of DNA where the break is made and the segment that is passed through the break, the net result is the removal of supercoils. If these two segments of DNA are on different molecules, catenation or decatenation results. These properties make topoisomerases required for essentially all macromolecular processes that operate on DNA in the cell (1-3).The basic sequence of events necessary for one round of topoisomerization has been outlined and incorporated into the two-gate model (4). Capture of a segment of DNA (the T segment) to be transported through the DNA break (the DNA gate) is accomplished by ATP binding-dependent dimerization of two halves of the enzyme (the N gate). This initiates a concerted series of events where the T segment is then forced through the DNA gate, which then closes, resulting in the passage of the T segment to the interior of the enzyme. Release of the T segment from the enzyme occurs upon opening of the C gate. ATP hydrolysis results in re-opening of the N gate, thereby resetting the enzyme for another cycle.The prokaryotic and eukaryotic enzymes share extensive amino acid sequence similarity and are organized in a similar fashion (5, 6). The eukaryotic enzymes are homodimers of a single polypeptide chain that contain a N-terminal ATP-binding domain and a C-terminal DNA cleavage domain. In the prokaryotic enzymes, these domains are on separate subunits and the protein is a heterotetramer.Electron microscopic analysis (7,8) and the solution of several crystal structures (4, 9, 10) have begun to provide a picture of the detailed conformational changes required for topoisomerization and have offered some insight to the mechanism of covalent catalysis and drug resistance. However, little is known about the regions of the protein required for coupling ATP-binding and hydrolysis to operation of the DNA gate.In order to define these regions and to detect regions of the ATP-binding subunit that are involved in cova...

Topoisomerase III Can Serve as the Cellular Decatenase in Escherichia coli

“…pBADtopB was constructed by inserting an NaeI-SmaI DNA fragment from pTBE302 (the gift of R. DiGate, University of Maryland) that contained araC as well as the topB ORF under the control of the ara promoter into the EcoRV site of pBR-kan-inc3. pLEXparE has been described previously (27).…”

“…3 and then chilled on ice. Superhelical DNA relaxation assays (27) and kinetoplast DNA decatenation (29) assays were then as described previously with 1 l of the heat-treated Topo IV being added to 20-l reaction mixtures. The extent of the reactions was quantitated using Bio-Rad Quantity One software from digital images of the ethidium bromide-stained gels.…”

Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli

“…Proteins were separated by SDS͞PAGE (24) and transferred to a poly(vinylidene difluoride) membrane (Micron Separations, Westboro, MA). Immunodetection was performed with polyclonal antibodies against ParC and ParE as the primary and goat anti-rabbit Ig G conjugated to horseradish peroxidase (New England Biolabs) as the secondary antibody (25). ECL substrate (Amersham Pharmacia) was used to detect the secondary antibody, and the ParC and ParE proteins were visualized by using Kodak X-Omat film.…”

“…To investigate whether the nature of the aberrant nucleoid morphology in cells lacking type I topoisomerase activity was caused by the accumulation of catenated chromosomes, we examined whether the overexpression of Topo IV could suppress the phenotype of these mutants. An isopropyl ␤-D-thiogalactoside (IPTG)-inducible Topo IV expression plasmid, plex5BA-parEC (25), was used to increase Topo IV activity within the cells. Plasmid plex5BA-parEC was introduced into DM750 ⌬topB containing plasmid pTBE33 (QZ107), a topB expression plasmid compatible with plex5BA-parEC.…”

Mutational Analysis of Escherichia coliTopoisomerase IV

“…We isolated six independent alleles in a screen for dominantnegative mutations in parC (7). The corresponding mutant ParE proteins all formed catalytically inactive Topo IV when reconstituted with ParC.…”