This chapter contains sections titled: Genomics and DNA Microarrays Toxicogenomics Challenges of Conventional Toxicology Approaches Opportunities for Genomics Processes and Methods for Toxicogenomics Experimental Design Data Quality Assessment Reference Compendium Generation Classification Diagnosis of Microarray Data Quality Sample Preparation Dye Incorporation Distortion Impurities Scanner Settings Automation of Data Quality Control Preprocessing of Microarray Data Generating a Reference Compendium of Compounds Cross Validation Mechanism of Action Alternative Structuring of Profiling Data Promoter Analysis Pathways Mapping Gene Expression Profiles onto Genomes In silico Comparative Genomics Outlook References
A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100–150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and reseal-ing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.
The sequence of the gyrase B subunit gene from Staphylococcus aureus strains resistant to the gyrase B subunit inhibitors cyclothialidine, coumermycin, and novobiocin has been determined. The residues altered in the resistant gyrase B subunits map to the ATP-binding region, suggesting that the drugs inhibit ATP binding and hydrolysis. The pattern of cross-resistances indicates that the detailed binding mode of the compounds differs.
The quinolones inhibit the A subunit of DNA gyrase in the presence of Mg2+ by interrupting the DNA breakage and resealing steps, and the latter step is also retarded without quinolones if Mg2+ is replaced by Ca2+. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca2+, we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca2+ was in addition dependent on the length of the DNA fragments.
The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [14C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [14C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [14C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2)·DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase·DNA complex (increase in affinity: B → A2B2 → A2B2·DNA). Furthermore, displacement curves of [14C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ. The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [14C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [14C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [14C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2)·DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase·DNA complex (increase in affinity: B → A2B2 → A2B2·DNA). Furthermore, displacement curves of [14C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ.
