Mutational Analysis of Escherichia coliTopoisomerase IV

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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...

“…Some of these amino acid residues have been mutated in other studies. Those results will be discussed in comparison to our observations as reported in the accompanying articles (12,13).…”

Section: Resultsmentioning

confidence: 49%

mentioning

confidence: 99%

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Mutational Analysis of Escherichia coliTopoisomerase IV

Mossessova¹,

Levine²,

Peng³

et al. 2000

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“…Each 30-l religation reaction contained an excess of enzyme over DNA by a factor 4 or 10; 3.6 nM negatively supercoiled pBR322 or 12.5 nM relaxed pBR322 was used with 33-50 nM of enzyme. In the first method, following previous reports (25,(27)(28)(29), the Ca 2ϩ -induced cleavage reactions were brought to 300 mM NaCl to initiate the religation reactions, which were incubated on ice for various times up to 20 min. The second method was a modification of the Mg 2ϩ -promoted religation described elsewhere (25,30).…”

Section: Methodsmentioning

confidence: 99%

The Action of the Bacterial Toxin Microcin B17

Pierrat¹,

Maxwell

2003

Microcins are a family of toxins produced by Enterobacteriaceae to inhibit phylogenetically related species (1). They differ from colicins by their smaller molecular mass and production at the stationary phase of growth (2). Microcin B17 (MccB17) 1 is a 3.1-kDa post-translationally modified peptide encoded on a plasmid in a single operon consisting of seven genes, mcbA to mcbG, responsible for synthesis, export, and immunity (3). Post-translational modifications involve both amino acid side chains and peptide backbones to form oxazole and thiazole rings (4 -6) that give MccB17 its characteristic structure (Fig. 1A).The action of microcin on sensitive Escherichia coli cells leads to an arrest of DNA replication and, consequently, to the induction of the SOS response (7). Moreover, MccB17 induces, in vitro and in vivo, double-stranded cleavage of DNA mediated by DNA gyrase (8). A mutation in the B subunit of gyrase (W751R) was found to confer resistance to MccB17, confirming that DNA gyrase is a cellular target of the toxin (8). MccB17 blocks gyrase by trapping the enzyme-DNA cleavage complex, a mode of action reminiscent of that of quinolones (8, 9). Of the two pairs of tandem, fused 4,2-bisheterocycles in MccB17, the integrity of the second one (B-site tandem; Fig. 1A) was found to be crucial for the potency of MccB17 (10, 11).DNA gyrase is a prokaryotic type II topoisomerase that negatively supercoils closed circular DNA with the requirement of ATP hydrolysis for energy (12, 13). The active enzyme is an A 2 B 2 heterotetramer with the A subunit (GyrA, 97 kDa) largely responsible for DNA wrapping and the breakage and reunion of DNA and the B subunit (GyrB, 90 kDa) responsible for ATP hydrolysis and the interaction with GyrA and DNA. The enzyme introduces supercoils into DNA by wrapping a doublestranded segment around itself, cleaving this DNA (the gate segment) in both strands, passing the wrapped DNA (transported segment) through the break, and then resealing the DNA (12-15). Binding of ATP to GyrB causes the closure of the clamp formed by dimerization of GyrB, which captures the transported segment and directs it through the doublestranded break in the gate segment. ATP is then hydrolyzed, allowing the enzyme to return to its starting conformation (16 -20).In addition to MccB17, other inhibitors of DNA gyrase include the antibacterial agents coumarins and quinolones and the bacterial toxin CcdB (21-23). Coumarins competitively inhibit ATP hydrolysis, whereas quinolones, such as ciprofloxacin (CFX) and oxolinic acid (OXO), stabilize the covalent gyrase-DNA cleavage complex. CcdB also stabilizes a cleavage complex but only in the presence of ATP (21). The trapping of the gyrase-DNA cleavage complex by MccB17 is also reported to be ATP-dependent (9). DNA cleavage complexes can also be observed in the absence of drugs or toxins, if Mg 2ϩ is substituted by Ca 2ϩ , with both gyrase (24) and eukaryotic topo II (25).The elucidation of the mode of action of MccB17 on DNA gyrase is important in at least two respects. F...

“…Because ParC binds DNA nearly as well as the intact enzyme, 2 this implies that ParE either contributes amino acid residues to the active site that are necessary for catalysis or that signals from ParE, in the form of conformational changes presumably driven by either the binding or hydrolysis of ATP, are required for DNA cleavage to proceed. As described in an accompanying article (6), Topo IV proteins reconstituted from ParC and mutant ParE proteins that are defective in either ATP binding or hydrolysis are still capable of covalent complex formation and DNA cleavage. This suggests that there must be other amino acid residues of ParE that are not involved in either ATP binding or hydrolysis, but are required for covalent catalysis.…”

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confidence: 99%

Mutational Analysis of Escherichia coliTopoisomerase IV

Bahng¹,

Mossessova²,

Nurse³

et al. 2000

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The products of three dominant-negative alleles of parE, encoding the ATP-binding subunit of topoisomerase IV (Topo IV), were purified and their activities characterized when reconstituted with ParC to form Topo IV. The ability of the ParE E418K, ParE G419D, and ParE G442D mutant Topo IVs to bind DNA, hydrolyze ATP, and close their ATP-dependent clamp was relatively unaffected. However, their ability to relax negatively supercoiled DNA was compromised significantly. This could be attributed to severe defects in covalent complex formation between ParC and DNA. Thus, these residues, which are far from the active site Tyr of ParC, contribute to covalent catalysis. This indicates that a dramatic conformational rearrangement of the protein likely occurs subsequent to the binding of the G segment at the DNA gate and prior to its opening.Type II topoisomerases are capable of passing segments of duplex DNA through transient double-strand breaks in a reaction that is coupled to ATP-binding and hydrolysis. When the passed segment (the T segment) and the transient doublestrand break (the DNA gate) are on the same circular DNA molecule, a change in the linking number of the DNA results. When the two segments are on two different DNA molecules, catenation or decatenation occurs. In prokaryotes, the ATP binding and hydrolysis and the DNA cleavage and religation activities reside on different subunits that associate to form a heterotetramer