Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.     

Investigation of the inhibitory role of phosphorothioate internucleotidic linkages on the catalytic activity of the restriction endonuclease EcoRV

The inhibitory effect of phosphorothioate residues, located within one strand of double-stranded DNA, on the hydrolytic activity of the restriction endonuclease EcoRV was investigated. Specific incorporation of a phosphorothioate group at the site of cleavage yielded the sequence 5'-GATsATC-3'. This modified sequence was cleaved at a relative rate of 0.1 compared to the unmodified substrate. Substrates 5'-GATsAsTC-3' and 5'-GsATsATC-3', both containing one additional phosphorothioate substitution, were linearized at a rate of 0.04 relative to unmodified DNA. However, under the same conditions, fully dAMPS-substituted DNA was found to be virtually resistant to the hydrolytic activity of EcoRV. Further experiments showed that double-stranded DNA fragments generated by PCR containing phosphorothioate groups within both strands are potent inhibitors of EcoRV catalysis. The inhibition was independent of whether the inhibitor fragment contained an EcoRV recognition site. We concluded that substitution of the phosphate group at the site of cleavage by a phosphorothioate residue decreases the rate of EcoRV-catalyzed hydrolysis most significantly. Substitution of other phosphate groups within the recognition sequence plays a limited role in enzyme inhibition. The presence of multiple dNMPS residues at regions of the DNA removed from the EcoRV recognition site may decrease the amount of enzyme available for catalysis by nonspecific binding to EcoRV.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.     

Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.

Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.     

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.     

Nonenzymatic sequence-specific cleavage of duplex DNA via triple-helix formation by homopyrimidine phosphorothioate oligonucleotides

Phenanthroline was attached covalently to the 5′-terminus of the unmodified and modified (3′-terminal phosphorothioate) oligonucleotide sequences, TTTTTTCTTCTCTTTCC (OP-17 mer) and TTTTTTTCTTCTCTTTCsC (OPRp-17 mer or OPSp-17 mer) via a phosphoramidite bond. Simian virus 40 DNA contains a single target site for these oligonucleotides. In the presence of copper ions, the efficient double-stranded cleavage at 37 °C and pH 7.0 was observed by agarose gel electrophoresis. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove, even though the linker was located in the major groove. Of particular interest are the 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp), which were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes and antisense nucleic acid based anti-viral therapies, which could be used as tools in cellular biology.

The 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp) were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes, which could be used as tools in cellular biology.     

DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage.

Escherichia coli DNA gyrase contains a 1:1 ratio of protomers coded by the genes gyrA and gyrB. This along with previous results shows that the enzyme has two copies of each protomer and thus a molecular weight of 400,000. Abortion of the gyrase reaction results in double-strand breakage of the DNA and covalent attachment of both gyrA protomers to the 5'-cut ends. We conclude that the gyrA protomer contains a critical part of the active site for the concerted breakage and reunion reaction of gyrase, the topoisomerase activity of the enzyme.     

DNA gyrase on the bacterial chromosome. Oxolinic acid-induced DNA cleavage in the dnaA-gyrB region

Oxolinic acid forms complexes with gyrase and DNA in such a way that subsequent denaturation of gyrase reveals DNA cleavage. Cleavage sites were mapped in a 10,000 base-pair region of the Escherichia coli chromosome containing the dnaA, dnaN, recF, and gyrB genes. Twenty-four cleavage sites were identified. The sites were cleaved at different frequencies, with the most frequent cleavage occurring within gyrB. Not all sites were equally sensitive to oxolinic acid concentration, some sites exhibited an altered cleavage frequency when the gyrB225 delta topA mutant strain DM800 was compared with wild-type cells, and coumermycin selectively changed the cleavage frequency at a few sites in the mutant strain DM800. These perturbations appear to alter the frequency of cleavage at a site but not the location of the site. The availability of many sites of differing strengths may be an important factor in the ability of gyrase to fine-tune the level of supercoiling or provide local swivels in bacterial DNA.     

Selective cleavage of pyrophosphate linkages.

Pyrophosphate linkages have a number of important roles in biology and are also formed chemically with great ease. They often are unwanted products, such as in the nonenzymatic oligomerization of mononucleotides. We have found that Zr(4+)- and Th(4+)-ions catalyze the symmetrical hydrolysis of pyrophosphate linkages. Oligonucleotide analogs linked by pyrophosphate bonds are substantially degraded in the presence of these metals, even at 0 degrees C. Conditions are described which permit the decapping of a pyrophosphate capped oligonucleotide. Oligodeoxynucleotides can be decapped by this procedure without cleavage of phosphodiester linkages. Oligoribonucleotides are susceptible to partial hydrolysis and require purification by HPLC after decapping.     

Comparison of the cleavage profiles of oligonucleotide duplexes with or without phosphorothioate linkages by using a chemical nuclease probe

A manganese porphyrin complex, Mn-TMPyP, associated with KHSO₅ is a chemical nuclease able to selectively recognize the minor groove of three consecutive AT base pairs of DNA and to mediate very precise cleavage chemistry at that particular site. This specific recognition and cleavage were used to probe the accessibility of the minor groove of DNA duplexes composed of one phosphodiester strand and one phosphorothioate strand. The cleavage of 5-GCAAAAGC/5-GCTTTTGC duplexes by Mn-TMPyP/KHSO₅ was monitored by HPLC coupled to electrospray mass analysis. Each single strand was synthesized with all-phosphate, all-Rp-phosphorothioate and all-Sp-phosphorothioate internucleotide bonds. We found that the manganese porphyrin was able to recognize its favorite (AT)₃-box binding site within the heteroduplexes, as in the case of natural DNA. Molecular modeling studies on the interactions of the reactive porphyrin manganese-oxo species with both types of duplexes confirmed the experimental data.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

DNA supercoiling by DNA gyrase

Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio of their concentrations. We established that the same linking number was attained independent of the direction from which the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present in the incubation, but remains a function of the [ATP]-to-[ADP] ratio. These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts for the experimental observations. According to this scheme our experimental results imply that there is significant slip in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling of DNA gyrase.     

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.     

Mechanism of illegitimate recombination: common sites for recombination and cleavage mediated by E. coli DNA gyrase

Summary Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.     

Locking the DNA gate of DNA gyrase: investigating the effects on DNA cleavage and ATP hydrolysis

Supercoiling by DNA gyrase involves the passage of one segment of double-stranded DNA through another. This requires a DNA duplex to be cleaved and the broken ends separated by at least 20 A. This is accomplished by the opening of a dimer interface, termed the DNA gate, which is covalently attached to the broken ends of the DNA. After strand passage, the DNA gate closes allowing the reunion of the broken ends. We have cross-linked the DNA gate of gyrase using cysteine cross-linking to block gate opening. We show that this locked gate mutant can bind quinolone drugs and perform DNA cleavage. However, locking the DNA gate prevents strand passage and the ability of DNA to stimulate ATP hydrolysis. We discuss the mechanistic implications of these results.     

Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase alpha.

The incorporation of cytosine arabinoside monophosphate (araCMP) into DNA at internucleotide linkages by DNA polymerase alpha (DNA pol alpha) has been investigated by using oligonucleotide primed DNA templates. The products of reactions catalyzed by DNA pol alpha in vitro were analyzed on polyacrylamide gels to measure insertion of araCMP, extension from an araCMP 3' terminus, and binding of the enzyme to an araCMP 3' terminus. The results show that insertion of araCMP opposite dGMP in the DNA template is about 3-fold less efficient than insertion of dCMP. Extension from an araCMP 3' terminus by addition of the next complementary nucleotide is approximately 2000-fold less efficient than extension from a correctly base-paired 3' terminus. In the absence of the second substrate, dNTP, DNA pol alpha binds with approximately equal affinities to DNA templates that contain oligonucleotide primers with araCMP or dCMP positioned at the 3' terminus. In the presence of dNTP, the enzyme extends the araCMP 3' terminus or dissociates, but it is not trapped at the araCMP 3' terminus in a nonproductive ternary complex as is observed at the ddCMP 3' terminus. To determine if slow phosphodiester bond formation contributes to the observed extension rate from the araCMP 3' terminus by DNA pol alpha, oligonucleotide primers with araCMP positioned at the 3' terminus were elongated by addition of the alpha-phosphorothioate analogue of the next complementary nucleotide. The rate of extension from araCMP by addition of 2'-deoxyadenosine 5'-O-phosphorothioate (dAMP alpha S) was 6-fold slower than by addition of dAMP, indicating that bond formation is partially rate limiting in the extension reaction. Thus, inefficient extension from the araCMP 3' terminus is the major determinant contributing to the low incorporation frequency of araCMP into DNA by DNA pol alpha, and this inefficiency can be attributed, in part, to slower phosphodiester bond formation at the araCMP 3' terminus.     

The binding of gyrase to DNA: analysis by retention by nitrocellulose filters.

Three distinct Escherichia coli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter-binding assay. One complex consists of an ensemble of two subunit A and two subunit B protomers bound noncovalently to specific sequences of DNA. High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter. At 23 degrees, the complex has a dissociation constant of approximately 10(-10) M and a half-time of decay of about 60 h. It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity. Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10. On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants. The first and second complexes require Mg++ for optimal formation. The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.     

Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli.

Escherichia coli DNA gyrase is comprised of two subunits, GyrA and GyrB. Previous studies have shown that GyrI, a regulatory factor of DNA gyrase activity, inhibits the supercoiling activity of DNA gyrase and that both overexpression and antisense expression of the gyrI gene suppress cell proliferation. Here we have analyzed the interaction of GyrI with DNA gyrase using two approaches. First, immunoprecipitation experiments revealed that GyrI interacts preferentially with the holoenzyme in an ATP-independent manner, although a weak interaction was also detected between GyrI and the individual GyrA and GyrB subunits. Second, surface plasmon resonance experiments indicated that GyrI binds to the gyrase holoenzyme with higher affinity than to either the GyrA or GyrB subunit alone. Unlike quinolone antibiotics, GyrI was not effective in stabilizing the cleavable complex consisting of gyrase and DNA. Further, we identified an 8-residue synthetic peptide, corresponding to amino acids (89)ITGGQYAV(96) of GyrI, which inhibits gyrase activity in an in vitro supercoiling assay. Surface plasmon resonance analysis of the ITGGQYAV-containing peptide-gyrase interaction indicated a high association constant for this interaction. These results suggest that amino acids 89--96 of GyrI are essential for its interaction with, and inhibition of, DNA gyrase.