Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage

Williams, Nicola L.; Howells, Alison J.; Maxwell, Anthony

doi:10.1006/jmbi.2001.4468

Cited by 65 publications

(54 citation statements)

References 45 publications

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“…Gyrase is known to have an intrinsic rate of ATP hydrolysis, which is generally low but is known to vary significantly depending on the GyrB preparation (40); the ATPase activity is stimulated by the presence of DNA (21). The results of ATP hydrolysis experiments (Fig.…”

Section: Resultsmentioning

confidence: 99%

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Heddle

Maxwell

2002

Antimicrob Agents Chemother

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107

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DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr 122 ) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp 426 3Asn) and GyrB(Lys 447 3Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolonebinding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.Quinolone drugs are a large and widely used class of synthetic antibacterial compounds (10,11,19). First-generation (acidic) quinolones include nalidixic acid and oxolinic acid (OXO). Subsequent generations have been modified to increase spectrum and potency. The most significant modification has been the addition of a fluorine atom at position C-6 in drugs such as ciprofloxacin (CFX), which results in a considerable increase in activity (30). The newer drugs also commonly contain a secondary amine in addition to the carboxylic acid group common to most quinolones, making them amphoteric rather than acidic (Fig. 1A). More recent modifications that increase drug potency include the presence of a methoxy group at C-8 (48).In Escherichia coli the major target for the quinolones is DNA gyrase (14,35). DNA gyrase is a type II topoisomerase that is able to alter the topological state of DNA by cleaving both strands, passing a double strand of DNA through the gap and resealing the ends (6). In the presence of ATP, this results in negative supercoiling, a reaction unique to gyrase. The crystal structures of the 43-kDa N-terminal domain of GyrB (responsible for ATP hydrolysis and capturing a DNA strand) and a 59-kDa fragment of the 64-kDa N-terminal domain of GyrA (containing the residues for DNA binding and cleavage and the quinolone resistance-determining region [QRDR; residues 67 to 106]) have been determined (24, 39). The structure of the 47-kDa C-terminal domain of GyrB is not known but can be inferred from the analogous enzyme from Saccharomyces cerevisiae, topoisomerase II, for which the crystal structures of a 92-kDa region have been solved (5, 12).Resistance to quinolones commonly arises in DNA gyrase via mutations in the QRDR (44). Such mutations include GyrA(Ser 83 3Trp), which gives Ϸ20-fold resistance to a wide range of quinolones (9, 46). This region of GyrA is close to t...

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Section: Resultsmentioning

confidence: 99%

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Heddle

Maxwell

2002

Antimicrob Agents Chemother

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107

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“…Protein-protein interaction can protect from proteolysis; for example, heterodimerization of the ␣ and ␥ subunits of a G protein protected the former from tryptic digestion (51). Evidence for N-terminal dimerization has been reported for E. coli MutL (28), yeast MutL␣ (35), and other members of the GHL superfamily, including Hsp90 (27) and bacterial gyrase B (26). We suggest that this dimerization occurs in the wild type and the single mutant forms as well, but the dimer intermediate of these forms is more transient.…”

Section: Discussionmentioning

confidence: 99%

Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair

Tomer¹,

Buermeyer²,

Nguyen³

et al. 2002

Journal of Biological Chemistry

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MutL␣, a heterodimer composed of Mlh1 and Pms2, is the major MutL activity in mammalian DNA mismatch repair. Highly conserved motifs in the N termini of both subunits predict that the protein is an ATPase. To study the significance of these motifs to mismatch repair, we have expressed in insect cells wild type human MutL␣ and forms altered in conserved glutamic acid residues, predicted to catalyze ATP hydrolysis of Mlh1, Pms2, or both. Using an in vitro assay, we showed that MutL␣ proteins altered in either glutamic acid residue were each partially defective in mismatch repair, whereas the double mutant showed no detectable mismatch repair. Neither strand specificity nor directionality of repair was affected in the single mutant proteins. Limited proteolysis studies of MutL␣ demonstrated that both Mlh1 and Pms2 N-terminal domains undergo ATP-induced conformational changes, but the extent of the conformational change for Mlh1 was more apparent than for Pms2. Furthermore, Mlh1 was protected at lower ATP concentrations than Pms2, suggesting Mlh1 binds ATP with higher affinity. These findings imply that ATP hydrolysis is required for MutL␣ activity in mismatch repair and that this activity is associated with differential conformational changes in Mlh1 and Pms2. Mismatch repair (MMR)1 helps to protect the genome from replication errors caused by DNA polymerases. Its pivotal role as caretaker of genome stability is exemplified by HNPCC, a hereditary predisposition to colon, rectal, and other cancers associated with germ line mutations in MMR (1).In Escherichia coli, the major players in the pathway are known to the extent that a full in vitro mismatch repair was reconstituted from purified components (2). A MutS homodimer recognizes and binds a mismatch and recruits a MutL homodimer to form an ATP-dependent ternary complex with DNA (3). MutL recruits and activates MutH (4), an endonuclease that introduces a specific nick on hemimethylated GATC sites, thus providing the strand discrimination signal required to ensure repair of the nascent strand. MutL also activates UvrD (5), a helicase that unwinds the duplex DNA from the nick, providing a single strand DNA substrate for an exonuclease. The repair pathway is bidirectional in that repair can initiate from GATC sites located either 5Ј or 3Ј to the mismatch (6). As a result, of the four exonucleases that have been found to participate in the pathway, two have 5Ј to 3Ј polarity, and the others have 3Јto 5Ј polarity (7). The single strand gap formed by the exonuclease is filled in by DNA polymerase III, and the nick is sealed by DNA ligase.The pathway in eukaryotes is less understood than in E. coli (reviewed in Refs. 8 and 9). Instead of one MutS, there are three MutS-like proteins that are involved in mutation avoidance, Msh2, Msh3, and Msh6. Msh2 and Msh6 associate to form MutS␣, the major MutS activity in MMR (reviewed in Ref. 9). Illustrating a similar level of complexity, there are four MutL homologs in mammals: Mlh1, Pms2 (closest to Pms1 in yeast), Pms1, and Mlh3....

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“…Our mutational studies combined with structural observations indicate that quinolones would preferentially bind to the ATP-bound conformation of topoisomerase IV. Although it has been suggested that quinolones might impair ATP binding to E. coli topoisomerase IV (3), limited proteolysis experiments and ATP binding and hydrolysis assays with the homologous E. coli gyrase clearly indicate that quinolones are able to bind the ATP-bound topoisomerase (35), which can still hydrolyze ATP, although at a lower rate (29,55). At least the destabilization of the dimer, the formation of which is necessary to bind ATP (8,54), would weaken the subunit interaction, resulting in a smaller number of productive topoisomerase IV-DNA complexes and finally in decreased susceptibility to quinolones.…”

Section: Vol 185 2003 Atp-bound Conformation Of Topoisomerase IV 6143mentioning

confidence: 99%

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).

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Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage

Cited by 65 publications

References 45 publications

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

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