Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (129K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Cove, M. E.
Right arrow Articles by Maxwell, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cove, M. E.
Right arrow Articles by Maxwell, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 1995 Oxford University Press 2716-2722

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs Matthew E. Cove, Andrew P. Tingey+ and Anthony Maxwell*

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK

Received May 7, 1997; Accepted May 30, 1997

ABSTRACT

We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a single-stranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to ~20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage.

INTRODUCTION

DNA gyrase is the enzyme from bacteria which can introduce supercoils into closed-circular DNA using the free energy of ATP hydrolysis (1 ,2 ). Gyrase is a member of a group of enzymes called DNA topoisomerases which are involved in the control of DNA topology (3 ). These enzymes can be divided into two types: type I enzymes catalyse reactions involving transient single-strand breaks in DNA while type II enzymes break both strands of the duplex. DNA gyrase is a type II enzyme and shares significant sequence similarity with other enzymes of the group (4 ). While many type II enzymes favour intermolecular topoisomerase reactions (e.g. decatenation), gyrase strongly favours intramolecular reactions and is the only enzyme able to catalyse the introduction of supercoils into DNA.

The mechanism of DNA supercoiling by gyrase involves the following steps: (i) the binding of the enzyme to DNA and the wrapping of a segment of DNA (~130 bp) around the enzyme in a positively-supercoiled sense; (ii) cleavage of this wrapped DNA (the G-segment) in both strands, involving the formation of covalent bonds between the 5'-phosphates at the break sites and specific tyrosines in the enzyme; (iii) passage of another segment of DNA (the T-segment) through this break; (iv) resealing of the break. Catalytic supercoiling requires the hydrolysis of ATP but in the presence of the non-hydrolysable ATP analogue ADPNP (5'-adenylyl [beta],[gamma]-imidodiphosphate) limited supercoiling can be achieved, indicating that nucleotide binding will promote one round of supercoiling and that hydrolysis is required for the enzyme to turnover.

DNA gyrase from Escherichia coli consists of two proteins, GyrA and GyrB, of molecular masses 97 and 90 kDa respectively; the active enzyme is an A2B2 complex. The gyrase proteins have been shown to be organised as functional domains (1 ,2 ). The GyrA protein consists of an N-terminal domain (59-64 kDa) which contains the active-site tyrosine (Tyr122) for DNA cleavage and has interactions with the quinolone drugs, and a C-terminal domain (33 kDa) involved in the wrapping of DNA around the A2B2 complex. The GyrB protein consists of an N-terminal domain (43 kDa) which hydrolyses ATP and binds the coumarin drugs, and a C-terminal domain (47 kDa) which binds to the A protein and DNA. The structure of the 43 kDa N-terminal domain complexed with ADPNP has been solved to 2.5 Å resolution by X-ray crystallography (5 ). The structure of a 59 kDa N-terminal fragment of GyrA has also recently been solved (6 ).

Gyrase is the target of a number of antibacterial agents many of which belong to the quinolone and coumarin classes (7 ). Coumarin drugs inhibit supercoiling by preventing the hydrolysis of ATP by gyrase (8 -10 ). The structure of an N-terminal sub-domain of GyrB (24 kDa) complexed with novobiocin has recently been determined (11 ). These data and recent binding experiments (12 ) support the idea that coumarins are competitive inhibitors of ATP binding. Quinolones (e.g. oxolinic acid and ciprofloxacin) inhibit DNA supercoiling by stabilising the complex between gyrase and cleaved DNA (8 ,9 ,13 ). Incubation of gyrase and DNA in the presence of a quinolone drug and denaturation of the complex leads to double-strand cleavage of the DNA with a 4-base stagger between the sites on the two strands. Specifically a tyrosyl-phosphate ester is formed between Tyr122 of GyrA and the 5'-phosphates of the protruding single-strand termini. The GyrA protein, which is covalently attached to the DNA, may be removed by digestion with proteinase K to reveal the broken DNA (14 ,15 ). DNA cleavage by gyrase can also be revealed in this way in the absence of quinolones when Ca2+ is substituted for Mg2+ in the reaction (16 ). Quinolone-induced DNA cleavage by gyrase has been shown to be reversible by treatment with EDTA or heat prior to the addition of denaturant (14 ,15 ). Although the supercoiling reaction of gyrase requires both subunits, DNA cleavage can be carried out by the N-terminal domain of GyrA complexed with GyrB (16 ,17 ); the smallest GyrA fragment which can carry out this reaction is ~59 kDa. Recently it has been shown that a 59 kDa N-terminal GyrA fragment complexed with GyrB can carry out ATP-dependent relaxation and decatenation in reactions reminiscent of more conventional type II enzymes (18 )


Figure 1.Oligonucleotides used in this study. The DNA sequence around the pBR322 990 site in M13-147 is given at the top and bottom of the figure. Oligonucleotides are shown as grey boxes which correspond in sequence to the bottom strand of M13-147. The major gyrase cleavage site in this region (25) is indicated.

The ability of quinolone drugs to trap a complex in which gyrase is covalently attached to broken DNA is strong evidence for the existence of a protein-DNA covalent intermediate in the supercoiling cycle. This reaction is common to all topoisomerases and a number of drugs can stabilise a cleavable complex in this way. In the case of eukaryotic topoisomerase II (topo II; the counterpart to gyrase in higher organisms), a number of anti-tumour drugs (e.g. etoposide, amsacrine) have been shown to stabilise such a complex (19 ,20 ).

Although the gyrase/quinolone cleavage reaction is well documented, there is relatively little known about its detailed mechanism. In the case of the eukaryotic enzymes (topo I and II) this reaction has been studied using suicide substrates. In this case short DNA molecules have been designed such that cleavage by the enzyme yields a product which cannot be religated, allowing the separation of the cleavage and religation half reactions (21 -24 ). In the case of gyrase this approach is not readily applicable, as the enzyme normally binds a stretch of ~130 bp with the cleavage site being approximately in the centre of the bound region (25 -29 ). However, it has recently been reported that, in the presence of the quinolone drugs ciprofloxacin (CFX) and fleroxacin, gyrase can cleave a 71 bp DNA fragment (30 ). This result is consistent with earlier observations of the stabilising effects of quinolones on the gyrase-DNA complex (31 ).

In this paper we have explored the use of short DNA fragments in the DNA gyrase cleavage reaction. Specifically we have cloned the preferred gyrase cleavage site from plasmid pBR322 (25 ,32 ,33 ) into bacteriophage M13 DNA and probed the DNA cleavage reaction by annealing oligonucleotides to the single-stranded form of this recombinant M13 DNA.

MATERIALS AND METHODS

Cloning

To construct M13-147, double-stranded M13mp18 DNA (34 ) was digested with SmaI and treated with calf-intestinal phosphatase [Boehringer Mannheim (35 )]. A 147 bp fragment containing the preferred gyrase cleavage site from pBR322 was excised from plasmid pSTD147 (36 ) by digestion with AvaI. The ends were blunted using Klenow DNA polymerase (New England BioLabs) and dNTPs and the fragment ligated with linearised M13mp18 using T4 DNA ligase (Gibco-BRL). The ligation mixture was transformed into competent XL1-Blue cells. The presence of a single copy of the 147 bp insert was confirmed by restriction enzyme analysis and by DNA sequencing.

Enzymes and DNA

The gyrase A and B proteins were purified as described by Hallett et al. (37 ). The 59 kDa N-terminal GyrA fragment (gift of Ms C.V. Smith) was prepared as described previously (17 ). Negatively-supercoiled and relaxed forms of plasmid pBR322 were prepared as described previously (38 ). Linear pBR322 was prepared by digestion of the supercoiled form with EcoRI. M13 double-stranded (ds) DNA was prepared as described by Sambrook et al. (35 ).

Single-stranded (ss) M13 DNA was prepared as described by Sambrook et al. (35 ) except that an additional purification step was introduced to separate the ssDNA from small quantities of sheared chromosomal E.coli DNA present. The DNA was resuspended in 10 mM Na2HPO4/NaH2PO4 buffer (pH 7.2) and applied to a 5 ml Econo-Pactm hydroxyapatite column (Bio-Rad). The column was washed with 10 mM phosphate buffer, and the ssDNA and dsDNA were eluted with a gradient of 10-500 mM phosphate buffer at the same pH. Single-stranded DNA eluted at a concentration of ~200 mM phosphate while the chromosomal DNA eluted at ~300 mM phosphate. The eluted samples were de-salted with NAP-25tm columns (Pharmacia) and the DNA precipitated with ethanol.

Oligonucleotides (oligos) were synthesised by Dr K.S. Lilley (University of Leicester) and repurified from polyacrylamide gels (35 ). Oligos were 5'-end labelled using T4 polynucleotide kinase (Gibco-BRL) and [[gamma]-32P]ATP in `1-phor-all plus' buffer (Pharmacia).

Enzyme assays

DNA supercoiling reactions were carried out as described previously (16 ), under the following conditions: 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 5 mM dithiothreitol, 6.5% (w/v) glycerol, 1.8 mM spermidine, 1.4 mM ATP, 0.36 mg/ml BSA, 9 [mu]g/ml tRNA, 10 [mu]g/ml relaxed pBR322 DNA; incubations were for 1 h at 25oC. DNA cleavage assays were performed in the presence of either 0.2 mM CFX or 1 mM oxolinic acid (OXO) under supercoiling reaction conditions with the exception of an incubation time of 45 min at 25oC. Reactions were stopped by the addition of 0.2% SDS and 0.1 mg/ml proteinase K and the incubation continued for 30 min at 37oC (16 ,17 ,39 ). Cleavage reactions in the presence of Ca2+ contained 4 mM CaCl2 in place of MgCl2. ATPase assays were carried out as described previously (40 ).

RESULTS

Construction of an M13 derivative containing a preferred gyrase cleavage site

In order to prepare ssDNA molecules containing a known gyrase cleavage site, a 147 bp DNA fragment from pBR322 containing the preferred quinolone-induced cleavage site, at bp 990 on the top strand, was cloned into M13. This site has been extensively studied previously (25 ,29 ,33 ,36 ). The M13 derivative was named M13-147.

The rates of supercoiling of relaxed M13-147 and M13 were compared in a time course with gyrase and found to be identical (data not shown), i.e. the introduction of an additional gyrase binding site made no significant difference to the rate of supercoiling. To determine the principal sites of gyrase cleavage in dsM13-147 and dsM13, an oxolinic acid-directed cleavage assay was carried out on the linear form of both substrates. From the sizes of the resulting products, it was possible to determine the approximate sites of gyrase cleavage. We found that the principal cleavage product in both cases originated from a site within M13, indicating that a strong gyrase cleavage site is already present in M13. However, the pattern of bands produced indicated that the cloned site was cleaved secondarily. Since M13 is about twice the size of pBR322, and given the weak sequence specificity of the gyrase cleavage reaction, it was not surprising to find that a strong cleavage site was already present in M13. The exact position of the strong cleavage site was not determined. Since the pBR322 `990' site has been extensively studied we continued to use this site for further work. This experiment indicates that the pBR322 990 site is not the strongest site for quinolone-induced gyrase cleavage; indeed other stronger sites are known to exist (41 -43 ).


Figure 2.Cleavage of partially double-stranded substrates by DNA gyrase (A2B2). Oligos were incubated with ssM13-147 in the presence of gyrase and either oxolinic acid or ciprofloxacin as indicated. Samples were analysed on 19% polyacrylamide gels under denaturing conditions. (A) Oligo F36; (B) oligos F26 and F28; (C) oligos F20, F22 and F24. In all experiments the size of the cleavage products were determined by comparison with several reference oligos; arrows indicate the position of one reference oligo in each panel.

When ssM13-147 was used as a substrate in CFX-induced gyrase cleavage reactions, no cleaved products were found. When ssM13-147 was used as a cofactor in gyrase ATPase reactions, no stimulation of the ATPase activity was seen, consistent with previous observations (44 ). By contrast, in the presence of dsM13-147, gyrase showed the expected level of DNA-dependent ATPase activity (data not shown). These results confirm that gyrase shows no detectable interaction with ssDNA.

Cleavage of partially dsDNA substrates

Having obtained an M13 derivative containing a preferred gyrase cleavage site in single-stranded form, it was now possible to anneal a range of oligonucleotides to the region containing the preferred site and determine whether they were cleavage substrates for gyrase. To show that such partial duplexes can be cleaved in this context, a 150-base oligo was made which spanned the entire 147 bp sequence. Using radiolabelled 150mer, cleavage stimulated by oxolinic acid and ciprofloxacin was shown to occur (data not shown). Figure 1 shows the range of shorter oligonucleotides used in subsequent experiments. In each case the labelled oligo was annealed to ssM13-147 (top strand) and used as a substrate for the gyrase cleavage reaction in the presence of oxolinic acid and ciprofloxacin. Using oligo R39 the conditions for the cleavage reaction were optimised. With 2.1 nM ssM13-147, 2-fold excess labelled oligo and excess gyrase (42 nM), we found that optimal levels of cleavage occurred with ~0.2 mM CFX and ~1 mM OXO. These levels of drug and enzyme are ~10-fold higher than those required with larger duplex substrates and may reflect a decreased stability of the complex between gyrase and the short double-stranded regions. As discussed below this is a likely consequence of weaker gyrase-DNA interactions in complexes in which there is interaction with only the central (cleaved) portion of the wrapped segment. During time-course experiments we found that maximal levels of cleavage occurred after ~30 min with the partially double-stranded substrates under these conditions.


Table 1 Cleavage of short DNA substrates by DNA gyrase
Oligonucleotide Cleavage of partial duplex substrates Cleavage of double-stranded substrates
  CFX OXO CFX OXO
F40 ++ ++ ++ ++
F40* ++ ++ ++ ++
L39 ++ ++ NT NT
R39 ++ ++ NT NT
F36 ++ ++ ++ ++
F34 ++ ++ ++ ++
F32 ++ ++ NT NT
F30 ++ ++ NT NT
F28 ++ ++ NT NT
F26 ++ ++ NT NT
F24 ++ ++ NT NT
F22 ++ ++ NT NT
F20 + + +a +a
F19 - - NT NT
F18 - - NT NT
F18* NT NT +a +a
F16 - - NT NT
F12 - - NT NT
++, cleavage; +, low-level cleavage; -, no cleavage; NT, not tested.
aIn these reactions a higher level of enzyme (120 nM) and longer incubation time (1.5 h) were used.

Gyrase cleavage reactions using the above conditions were carried out with the oligos shown in Figure 1 . Example autoradiographs of the results from these cleavage experiments are shown in Figure 2 and the results are summarised in Table 1 . We found that all oligos >20 bases were cleaved at a similar level by gyrase (Fig. 2 ). F20 was cleaved less efficiently, and F19, F18 and F18* showed little or no cleavage. Smaller oligos (F16 and F12) showed no detectable cleavage. Although the site of cleavage was similar with all oligos there was some variation (Fig. 3 ), with the predominant cleavage site being different for the two drugs. In the presence of OXO the major cleavage site was at the expected 990 position, but with CFX the major site was 4 bases away at base 994 on the top strand (Fig. 3 ). In the case of CFX a number of secondary cleavage sites were also noted. It was found that cleavage would not occur when the site was <= 6 bp from the end of a fragment. For example, with F26 the major CFX site is no longer cleaved as it is now 6 bp away from the end of the oligo. Six different CFX cleavage sites around the 990 site have been observed in this study (Fig. 3 ), four of which are between GG dinucleotides in one of the strands (see Discussion). Although it seems that gyrase can cleave DNA duplexes down to ~20 bp in size in the presence of quinolones, we were unable to detect cleavage stimulated by Ca2+; for example R39 shows no detectable cleavage products in the presence of Ca2+. This suggests important differences between the stabilisation of the cleavable complex by quinolones and Ca2+.

It is feasible that the results with the partially double-stranded substrates might be influenced by flanking sequences, i.e. if the single-stranded part of M13-147 helps to stabilise the gyrase complex. Therefore selected cleavage reactions were carried out using double-stranded versions of the oligonucleotides (Table 1 ). We found that the results were consistent with those obtained using the partially double-stranded substrates.

Cleavage reactions with the 59 kDa N-terminal domain of GyrA

The DNA cleavage activity of DNA gyrase has been shown to reside within the N-terminal domain of GyrA (16 ) and fragments derived from the N-terminal part of GyrA, when complexed with GyrB, have been shown to cleave DNA in the presence of quinolones as efficiently as the intact enzyme (17 ). Using selected oligos (down to F24) we found that the cleavage reaction was identical to that with the intact enzyme (e.g. Fig. 4 ). This result implies that with these substrates the 33 kDa domain of GyrA, which is involved in DNA wrapping (45 ), does not influence the cleavage reaction and that, in these experiments, we are observing the interaction between DNA and the DNA breakage-reunion domain of GyrA (see Discussion).


Figure 3.Summary of cleavage sites. Thick arrows indicate major sites, thin arrows indicate minor sites. Starred sites were only observed in fragment R39. Sites on the bottom strand were directly determined from autoradiographs (e.g. Fig. 2); top strand sites are inferred. The position of oligo F18* (the smallest oligo which could be cleaved) is indicated.

Suicide substrates

A fruitful approach to probing the DNA cleavage reactions of topoisomerases and other enzymes has been the use of `suicide' substrates (21 ,46 ,47 ). These substrates are constructed such that, following cleavage, religation is disfavoured so that cleaved intermediates can be isolated. With eukaryotic topo II, suicide substrates can be made with short oligonucleotides (e.g. 17 bp) where the cleavage site is close to one end such that cleavage releases a short single-stranded segment [e.g. 3 bases (21 )]. Generally such an approach is unfeasible with gyrase as it normally binds ~130 bp of DNA. However, the observations made here on the cleavage of duplexes down to ~20 bp in size now makes this approach possible. We therefore constructed a number of suicide substrates based around F36 (Fig. 1 ) such that cleavage at either the OXO or major CFX site would release a short single-stranded DNA segment. Although we constructed substrates with pre-existing nicks close to the expected cleavage sites (e.g. 2 bases to the 3'-side) we found that cleavage would not occur within 6 bp of the nick, consistent with the observations made above. Attempts to disrupt the cleavage-religation process with nicked substrates, by for example including a heating step (to 65oC), failed to reveal any cleavage products.

DISCUSSION

In order to probe the DNA cleavage reaction of DNA gyrase we have cloned the preferred quinolone-induced cleavage site from plasmid pBR322 into M13. We have shown that in its single-stranded form this modified M13 does not appear to interact with DNA gyrase. We have prepared a series of complementary oligonucleotides, between 12 and 150 bases long, which anneal to the region containing the preferred cleavage site, and have assessed the ability of gyrase to cleave these partial duplex substrates in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Surprisingly, we find that in the presence of both drugs the enzyme can cleave substrates down to ~20 bp in size. In other work (e.g. footprinting and ATPase experiments) it was found that gyrase interacts with DNA duplexes of >= 100 bp (25 -29 ,48 ). However, drug-induced cleavage with the quinolone fleroxacin has been found with a 71 bp molecule (30 ), although oxolinic acid-induced cleavage of a 34 bp duplex, based on the pBR322 990 site (33 ), was found not to occur. We find that a very similar 34 bp substrate and indeed smaller substrates (Table 1 ) are in fact cleaved. These differences may be due to differing reaction conditions. Gmünder et al. (49 ) have also recently shown that gyrase can cleave DNA duplexes as short as 20 bp in the presence of quinolone drugs.


Figure 4.Cleavage of partially double-stranded substrates by A592B2. Cleavage of F24 and F40 by A592B2 in the presence of oxolinic acid or ciprofloxacin as indicated. Samples were analysed as described in the legend to Figure 2.

We found that DNA cleavage stimulated by Ca2+ did not occur with the short duplexes (<39 bp), consistent with the work of Gmünder et al. who showed that Ca2+ cleavage did not occur within an 85 bp fragment (30 ), but could be observed with fragments >96 bp (49 ). It has also been shown that Ca2+-induced cleavage of linear DNA is not catalysed by the N-terminal 64 kDa domain of GyrA (in the presence of GyrB) unless the C-terminal 33 kDa wrapping domain is also present (50 ). Again these results suggest important differences in the quinolone- and Ca2+-induced cleavage of DNA by gyrase.

It is well known that when gyrase binds DNA a section of ~130 bp is wrapped around the enzyme. This consists of a central portion of 13-40 bp (25 -29 ) and flanking regions. It is likely that the central portion interacts with the N-terminal domain of GyrA containing the DNA breakage-reunion site whilst the flanking portions interact with the 33 kDa C-terminal `wrapping' domain of GyrA. This is borne out by recent experiments which shows that the complex between the 59 kDa N-terminal domain of GyrA and GyrB shows no DNA wrapping (18 ). In addition, transcriptional footprinting of gyrase in the presence of CFX indicates that ~20 bp of DNA surrounding the 990 cleavage site is inaccessible to T7 RNA polymerase (51 ). Thus, we suggest that the binding of gyrase to DNA consists of two elements, a central segment within which DNA cleavage occurs and flanking regions involved in DNA wrapping. It would appear that, for quinolone-induced cleavage, binding to only the central portion is required. It is interesting to note that the limit size of DNA fragments cleavable by gyrase is very similar to the size of fragments that can be cleaved by topo II. For example Lund et al. (52 ) have shown that calf thymus topo II can cleave a duplex of 16 bp. This is also consistent with the fact that when topo II binds DNA it cleaves but does not wrap and therefore has a correspondingly smaller footprint than gyrase [~25 bp (53 )].

Although we found that cleavage induced by oxolinic acid occurred at the expected 990 site, cleavage induced by ciprofloxacin occurred at a number of positions with 994 being the preferred site (Fig. 3 ). It is likely that cleavage specificity in DNA gyrase will be determined by a number of factors. Firstly, interactions of the enzyme with the DNA bases surrounding the cleavage site, secondly, interaction of drug molecules with DNA bases around the cleavage site, and thirdly, the positioning effect determined by the wrapping of DNA around the 33 kDa domains. In this paper wrapping is clearly not involved and this is indeed borne out by the similarity in the cleavage reaction of intact gyrase and that of the 59 kDa domain (Fig. 4 ). In earlier work using longer DNA molecules (147 and 198 bp) drug-induced cleavage was found to occur almost exclusively at the 990 site (29 ,36 ). This suggests that the DNA wrap, mediated by the 33 kDa domains of GyrA, exerts a preferred rotational orientation on the bound DNA and thus prescribes the site of cleavage. It is likely that preferred sites are those where the flanking DNA has an intrinsic curvature or higher than average flexibility. In the experiments described in this paper the constraints imposed by DNA wrapping are not present and the short fragments are free to bind in the enzyme's active site in any orientation. Hence a range of cleavage sites is likely to arise with such fragments, as seen in Figure 3 . We suggest therefore that in these experiments cleavage sites may be determined largely by drug-DNA interactions.

In the work of Gmünder et al. (49 ), the principal cleavage sites with fleroxacin and ciprofloxacin were found to be at bp 990 and 993 in the top strand, compared with 990 and 994 in our work (Fig. 3 ). It is not clear why these sites are not identical but it may reflect different reaction conditions and the variability of cleavage sites with fluoroquinolones. It is worth noting in this regard that in previous work by Gmünder et al. (30 ) quinolone-induced cleavage by gyrase of DNA fragments based on the 990 site occurred at 990 and 994. In this work cleavage of the top strand was monitored which supports the assertion that the cleavage sites we have observed are indeed double-stranded breaks with a 4-base stagger between sites on opposite strands (see below).

In the experiments described in this paper only bottom strand cleavage was generally followed, the bottom strand being the labelled oligo and the top strand being intact single-stranded M13-147 (Fig. 3 ). The assumption that cleavage also occurred in the top strand at the expected locations is supported by several considerations. Firstly, cleavage of DNA by gyrase to generate a double-stranded break with a 4-base stagger has been widely observed (25 ,26 ,54 ,55 ). Secondly, the two major sites we observe in the bottom strand (990 and 994) have also been independently observed in the top strand by Gmünder et al. (30 ). Thirdly, we have carried out control experiments using double-stranded F28 labelled in either the top or bottom strand and observed cleavage products from both strands (data not shown).

The cleavage sites favoured by CFX seemed to show some sequence preferences. Looking at the bases either side of the point of cleavage in the top and bottom strands in the six CFX sites near 990 (Fig. 3 ), there seems to be a preference for G bases. All sites have a purine-purine dinucleotide in at least one strand, and in four out of the six sites cleavage occurs between a GG dinucleotide in one strand. Previous work has suggested a preference for quinolone binding to G residues and it is possible that the data in this paper also reflect this preference for binding to Gs, perhaps indicative of drug-DNA base interaction. For example, nalidixic acid has been shown to bind to guanine on ssDNA in the presence of copper ions (56 ,57 ), and norfloxacin showed a preference for single-stranded G-containing homopolymers (58 ). Gmünder et al. (49 ) found that gyrase-catalysed cleavage in the presence of fleroxacin and CFX showed a preference for a G residue 5' to the cleavage site in at least one strand. However, a detailed study of sequence preferences would require the analysis of a number of different gyrase cleavage sites.

Another interesting feature of drug-induced cleavage from this work is that cleavage fails to occur at sites which are within 6 bp of the end of a duplex (Fig. 3 ). This might suggest that there is an important enzyme-DNA contact, ~6-7 bases away from the cleavage site, which is required for cleavage. Therefore, in theory, gyrase should be capable of cleaving a duplex which is 18 bp in length, with the cleavage site in the centre. In support of this suggestion we find that F18* is cleaved weakly by gyrase at the site in the centre of the duplex region (Fig. 1 and Table 1 ). It is therefore unlikely that gyrase would cleave a duplex region <18 bp in length, because cleavage would have to occur within 6 bases of either end of the duplex. This is distinct from eukaryotic topo II which can cleave a substrate with only 3 bp on one side of the cleavage site (21 ).

In the work of Gmünder et al. (49 ) a ladder of products formed by cleavage close to the ends of their substrate DNAs was found. The formation of these products was Mg2+ dependent and the activity was present with GyrA or GyrB alone. The authors speculate that this activity is due to non-specific cleavage activity of gyrase or the presence of a co-purifying nuclease. Our work tends to favour the latter explanation. Our initial experiments also revealed ladders of cleavage products which we found to be due to a Mg2+-dependent activity associated with GyrB. As our GyrB had no intrinsic supercoiling activity it was unlikely to be contaminated with GyrA. We found that the laddering could be substantially reduced by using an altered purification procedure involving a GyrA-affinity column (59 ). Evidence presented above also argues against gyrase cleaving at the ends of DNA substrates because of the requirement for at least 6 bp on each side of the cleavage site.

One of the aims of this work was to develop substrates suitable for suicide cleavage reactions. The observations of cleavage of short duplexes suggested that this approach might be feasible. However, although a number of suicide substrates were designed based around F36 and a number of cleavage conditions explored, we were unable to find evidence of suicide reactions. [Failure of gyrase to cleave suicide substrates has also been noted in another lab (H. Gmünder, personal communication)]. One reason for this is the requirement for at least 6 bases either side of the cleavage site, discussed above. The fact that substrates with >6 bases either side but containing a nick within the 6 bases were not cleaved might imply that the requirement reflects a structural transition in the DNA which occurs at the cleavage site. The recently determined crystal structure of the 59 kDa N-terminal domain of GyrA (6 ) suggests that distortion of the DNA near the cleavage site is likely to occur, i.e. the presence of a nick near the cleavage site may prevent distortion of the DNA required for cleavage.

In summary, these experiments show that gyrase can cleave DNA duplexes down to ~20 bp in size but cannot cleave at a site which is <6 bp from the end of a fragment. The quinolone drug CFX shows preferences for G bases adjacent to the cleavage site, which may reflect drug-base interaction.

ACKNOWLEDGEMENTS

We thank Hans Gmünder and Sotirios Kampranis for helpful comments. MEC acknowledges Knoll Pharmaceuticals (formerly Boots) and the Leicestershire Clinical Research Committee for financial support, and APT acknowledges BBSRC and Glaxo-Wellcome for a CASE studentship. AM is a Lister Institute Jenner Fellow.

REFERENCES

1 Reece, R.J. and Maxwell, A. (1991) CRC Crit. Rev. Biochem. Mol. Biol., 26, 335-375.

2 Wigley, D.B. (1995) In Eckstein, F. and Lilley, D.M.J. (eds), Nucleic Acids and Molecular Biology volume 9. Springer-Verlag, Berlin, Heidelberg, pp. 165-176.

3 Wang, J.C. (1996) Annu. Rev. Biochem., 65, 635-692. MEDLINE Abstract

4 Caron, P.R. and Wang, J.C. (1994) Adv. Pharmacol., 29B, 271-297.

5 Wigley, D.B., Davies, G.J., Dodson, E.J., Maxwell, A. and Dodson, G. (1991) Nature, 351, 624-629. MEDLINE Abstract

6 Morais Cabral, J.H., Jackson, A.P., Smith, C.V., Shikotra, N., Maxwell, A. and Liddington, R.C. (1997) Nature, in press.

7 Maxwell, A. (1997) Trends Microbiol., 5, 102-109. MEDLINE Abstract

8 Drlica, K. and Coughlin, S. (1989) Pharm. Ther., 44, 107-121.

9 Rádl, S. (1990) Pharm. Ther., 48, 1-17.

10 Maxwell, A. (1993) Mol. Microbiol., 9, 681-686. MEDLINE Abstract

11 Lewis, R.J., Singh, O.M.P., Smith, C.V., Skarynski, T., Maxwell, A., Wonacott, A.J. and Wigley, D.B. (1996) EMBO J., 15, 1412-1420. MEDLINE Abstract

12 Gormley, N.A., Orphanides, G., Meyer, A., Cullis, P.M. and Maxwell, A. (1996) Biochemistry, 35, 5083-5092. MEDLINE Abstract

13 Maxwell, A. (1992) J. Antimicrob. Chemother., 30, 409-416. MEDLINE Abstract

14 Gellert, M., Mizuuchi, K., O'Dea, M.H., Itoh, T. and Tomizawa, J. (1977) Proc. Natl. Acad. Sci. USA, 74, 4772-4776. MEDLINE Abstract

15 Sugino, A., Peebles, C.L., Kruezer, K.N. and Cozzarelli, N.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 4767-4771. MEDLINE Abstract

16 Reece, R.J. and Maxwell, A. (1989) J. Biol. Chem., 264, 19648-19653. MEDLINE Abstract

17 Reece, R.J. and Maxwell, A. (1991) J. Biol. Chem., 266, 3540-3546. MEDLINE Abstract

18 Kampranis, S.C. and Maxwell, A. (1996) Proc. Natl. Acad. Sci. USA, 93, 14416-14421. MEDLINE Abstract

19 Chen, A.Y. and Liu, L.F. (1994) Annu. Rev. Pharmacol. Toxicol., 34, 191-218. MEDLINE Abstract

20 Froelich-Ammon, S.J. and Osheroff, N. (1995) J. Biol. Chem., 270, 21429-21432. MEDLINE Abstract

21 Andersen, A.H., Sørensen, B.S., Christiansen, K., Svejstrup, J.Q., Lund, K. and Westergaard, O. (1991) J. Biol. Chem., 266, 9203-9210.

22 Svejstrup, J.Q., Christiansen, K., Gromova, I.I., Andersen, A.H. and Westergaard, O. (1991) J. Mol. Biol., 222, 669-678. MEDLINE Abstract

23 Sørensen, B.S., Sinding, J., Andersen, A.H., Alsner, J., Jensen, P.B. and Westergaard, O. (1992) J. Mol. Biol., 228, 778-786. MEDLINE Abstract

24 Schmidt, V.K., Sørensen, B.S., Sørensen, H.V., Alsner, J. and Westergaard, O. (1994) J. Mol. Biol., 241, 18-25. MEDLINE Abstract

25 Fisher, L.M., Mizuuchi, K., O'Dea, M.H., Ohmori, H. and Gellert, M. (1981) Proc. Natl. Acad. Sci. USA, 78, 4165-4169. MEDLINE Abstract

26 Kirkegaard, K. and Wang, J.C. (1981) Cell, 23, 721-729. MEDLINE Abstract

27 Morrison, A. and Cozzarelli, N.R. (1981) Proc. Natl. Acad. Sci. USA, 78, 1416-1420. MEDLINE Abstract

28 Rau, D.C. , Gellert, M., Thoma, F. and Maxwell, A. (1987) J. Mol. Biol., 193, 555-569. MEDLINE Abstract

29 Orphanides, G. and Maxwell, A. (1994) Nucleic Acids Res., 22, 1567-1575. MEDLINE Abstract

30 Gmünder, H., Kuratli, K. and Keck, W. (1995) Antimicrob. Agents Chemother., 39, 163-169. MEDLINE Abstract

31 Higgins, N.P. and Cozzarelli, N.R. (1982) Nucleic Acids Res., 10, 6833-6847. MEDLINE Abstract

32 Lockshon, D. and Morris, D.R. (1985) J. Mol. Biol., 131, 63-74.

33 Fisher, L.M., Barot, H.A. and Cullen, M.E. (1986) EMBO J., 5, 1411-1418. MEDLINE Abstract

34 Yanisch-Perron, C.J., Viera, J. and Messing, J. (1985) Gene, 33, 103-119.

35 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

36 Dobbs, S.T., Cullis, P.M. and Maxwell, A. (1992) Nucleic Acids Res., 20, 3567-3573. MEDLINE Abstract

37 Hallett, P., Grimshaw, A.J., Wigley, D.B. and Maxwell, A. (1990) Gene, 93, 139-142. MEDLINE Abstract

38 Bates, A.D. and Maxwell, A. (1989) EMBO J., 8, 1861-1866. MEDLINE Abstract

39 Tamura, J.K., Bates, A.D. and Gellert, M. (1992) J. Biol. Chem., 267, 9214-9222. MEDLINE Abstract

40 Ali, J.A., Jackson, A.P., Howells, A.J. and Maxwell, A. (1993) Biochemistry, 32, 2717-2724. MEDLINE Abstract

41 Wahle, E. and Kornberg, A. (1988) EMBO, 7, 1889-1895.

42 Yang, Y. and Ferro-Luzzi Ames, G. (1988) Proc. Natl. Acad. Sci. USA, 85, 8850-8854. MEDLINE Abstract

43 Pato, M.L., Howe, M.M. and Higgins, N.P. (1990) Proc. Natl. Acad. Sci. USA, 87, 8716-8720. MEDLINE Abstract

44 Sugino, A. and Cozzarelli, N.R. (1980) J. Biol. Chem., 255, 6299-6306. MEDLINE Abstract

45 Reece, R.J. and Maxwell, A. (1991) Nucleic Acids Res., 19, 1399-1405. MEDLINE Abstract

46 Svejstrup, J.Q., Christiansen, K., Andersen, A.H., Lund, K. and Westergaard, O. (1990) J. Biol. Chem., 265, 12529-12535. MEDLINE Abstract

47 Burgin, A.B., Huizenga, B.N. and Nash, H.A. (1995) Nucleic Acids Res., 23, 2973-2979. MEDLINE Abstract

48 Maxwell, A. and Gellert, M. (1984) J. Biol. Chem., 259, 14472-14480. MEDLINE Abstract

49 Gmünder, H., Kuratli, K. and Keck, W. (1997) Nucleic Acids Res., 25, 604-611. MEDLINE Abstract

50 Critchlow, S.E. and Maxwell, A. (1996) Biochemistry, 35, 7387-7393. MEDLINE Abstract

51 Willmott, C.J.R., Critchlow, S.E., Eperon, I.C. and Maxwell, A. (1994) J. Mol. Biol., 242, 351-363.

52 Lund, K., Andersen, A.H., Christiansen, K., Svejstrup, J.Q. and Westergaard, O. (1990) J. Biol. Chem., 265, 13856-13863. MEDLINE Abstract

53 Lee, M.P., Sander, M. and Hsieh, T. (1989) J. Biol. Chem., 264, 21779-21787. MEDLINE Abstract

54 Morrison, A. and Cozzarelli, N.R. (1979) Cell, 17, 175-184. MEDLINE Abstract

55 Gellert, M., Fisher, L., Ohmori, H, O'Dea, M. and Mizuuchi, K. (1980) Cold Spring Harbor Symp. Quant. Biol., 45, 391-398.

56 Crumplin, G.C., Midgley, J.M. and Smith, J.T. (1980) In Sammes, P.G. (ed), Topics in Antibiotic Chemistry, volume 3. Ellis Harwood Ltd, Chichester, pp. 13-35.

57 Dreyfuss, M.E. and Midgley, J.M. (1983) J. Pharm. Pharmacol., 35, 75P.

58 Shen, L.L., Baranowski, J. and Pernet, A.G. (1989) Biochemistry, 28, 3879-3885. MEDLINE Abstract

59 Maxwell, A. and Howells, A.J. (1997) In Bjornsti, M.-A. and Osheroff, N. (eds), Protocols for DNA Topoisomerases I. DNA topology and Enzyme Purification. Humana Press, Towata, New Jersey.

*To whom correspondence should be addressed. Tel: +44 116 252 3464; Fax: +44 116 252 3369; Email: ony@leicester.ac.uk

+Present address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
B. P. Belotserkovskii, P. B. Arimondo, and N. R. Cozzarelli
Topoisomerase Action on Short DNA Duplexes Reveals Requirements for Gate and Transfer DNA Segments
J. Biol. Chem., September 1, 2006; 281(35): 25407 - 25415.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
C. G. Noble, F. M. Barnard, and A. Maxwell
Quinolone-DNA Interaction: Sequence-Dependent Binding to Single-Stranded DNA Reflects the Interaction within the Gyrase-DNA Complex
Antimicrob. Agents Chemother., March 1, 2003; 47(3): 854 - 862.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
D. Strumberg, J. L. Nitiss, J. Dong, J. Walker, M. C. Nicklaus, K. W. Kohn, J. G. Heddle, A. Maxwell, S. Seeber, and Y. Pommier
Importance of the Fourth Alpha-Helix within the CAP Homology Domain of Type II Topoisomerase for DNA Cleavage Site Recognition and Quinolone Action
Antimicrob. Agents Chemother., September 1, 2002; 46(9): 2735 - 2746.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
J. Heddle and A. Maxwell
Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB
Antimicrob. Agents Chemother., June 1, 2002; 46(6): 1805 - 1815.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
M. L. Pato and M. Banerjee
Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication
J. Bacteriol., September 15, 1999; 181(18): 5783 - 5789.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
S. C. Kampranis and A. Maxwell
The DNA Gyrase-Quinolone Complex. ATP HYDROLYSIS AND THE MECHANISM OF DNA CLEAVAGE
J. Biol. Chem., August 28, 1998; 273(35): 22615 - 22626.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (129K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Cove, M. E.
Right arrow Articles by Maxwell, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cove, M. E.
Right arrow Articles by Maxwell, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?