Interactions between single‐stranded DNA‐binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging‐strand DNA polymerase. Here, the SSB‐binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging‐strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt‐dependent uncoupling of leading‐ and lagging‐strand DNA synthesis and to a surprising obstruction of the leading‐strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature‐dependent cell cycle defects that appear to arise from replisome instability.

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MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation exhibit nucleoid decondensation.

Edited by Patrick SungProperly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.The Escherichia coli chromosome is condensed about 1000-fold to fit into the nucleoid of the bacterium. A number of factors contribute to this extreme DNA condensation, among them are: DNA supercoiling, the binding of various nucleoid associated proteins like HU, Fis, and H-NS, and the binding of the structural maintenance of chromosomes (SMC) 3 -like condensin MukBEF (1, 2). SMC proteins act to manage the shape and behavior of chromosomes in both prokaryotes and eukaryotes (3). These proteins dimerize at a hinge region that is flanked by long coiled-coil regions that can be 40 -50 nm in length and that end in head domains that bind and hydrolyze ATP, as well as the bridging kleisin protein (MukF for the E. coli condensin (4)). The kleisin is then itself bound by another protein (MukE for the E. coli condensin (4)). There are three versions of the eukaryotic SMC proteins: the condensin, required for packaging of the chromosomes and comprised of SMC2 and SMC4; the cohesin, required for holding sister chromosomes together during mitosis and comprised of SMC1 and SMC3; and the SMC5-SMC6 complex, required for various aspects of DNA repair (3).It is generally acknowledged that the eukaryotic condensin and cohesin trap DNA topologically in the protein triangle formed by the SMC proteins and the kleisin (5, 6), although an alternative model for DNA binding for the eukaryotic cohesin exists (7). Recent reports suggest that the Bacillus subtilis SMC protein (8) and MukB (9) also trap chromosomes topologically. MukB binds linear and circular DNA in vitro and can induce negative supercoils and knots in relaxed circular DNA in the presence of a topoisomerase (10). Binding of MukB to chromosomal DNA in vivo requires ATP and MukEF (11, 12), although MukB DNA binding in vitro does not (10, 13).We (14) and the Burger and Oakley (15) labs have shown that the MukB hinge region interacts with the C-terminal ␤-propeller region of the ParC subunit of the cellular decatenase topoisomerase IV (Topo IV). We have reported (16) that this interaction stimulates the intramolecular activities of Topo IV, negative supercoil relaxation and knotting, but not the intermolecular activities of Topo IV, catenation/decatenation of DNA rings; whereas Berger, Oakley and colleag...

The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation.

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...

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein–DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.

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.Type II topoisomerases couple the energy of ATP hydrolysis to alter the linking number of DNA (1-3). 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. Thus, these enzymes have two catalytic activities: ATP binding and hydrolysis, and DNA cleavage and religation.Roca and Wang (4) clearly demonstrated that one role for ATP binding and hydrolysis is the operation of a protein clamp on the enzyme to trap the segment of DNA (the T segment) to be transported through the transient double-strand break (the G segment). Use of such a clamp is probably not an absolute necessity for topoisomerase activity, but it clearly increases the catalytic efficiency of the enzyme by orders of magnitude. ATP binding is sufficient to close the clamp, whereas hydrolysis of ATP is presumed to reset it in the open position, although this has not been demonstrated directly.DNA cleavage and religation functions to open and close the DNA gate through which the T segment must be passed in order to effect changes in DNA topology (1-3). Both DNA cleavage and religation and ATP binding and hydrolysis are manifest in the absence of the other activity, although DNA binding appears to stimulate ATP hydrolysis (1-3). Yet, it is clear that, during topoisomerization, the two reactions must be coordinated. ATP hydrolysis without DNA transport would render the enzyme very inefficient, allowing the captured T segment to escape without alteration of DNA topology.Little is known about the manner in which these reactions are coordinated and the amino acid residues important for this crucial linking of enzymatic functions. 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 (Top...