A 38-year-old woman, who works as an administrative assistant for a large company, opened a package and found a suspicious white powder. Analysis of the powder indicates that it contained traces of the bacterium Bacillus anthracis. The woman was treated with ciprofloxacin, an effective antibiotic. Ciprofloxacin’s mechanism of action is best described as an inhibition of which of the following?
A. Bacterial dihydrofolate reductase
B. Bacterial peptidyl transferase activity
C. Bacterial RNA polymerase
D. DNA gyrase
E. DNA polymerase III
The correct answer is D- DNA gyrase.
The quinolones, including nalidixic acid and its fluorinated derivatives (ciprofloxacin, levofloxacin, and moxifloxacin), are synthetic compounds that inhibit the activity of the A subunit of the bacterial enzyme DNA gyrase as well as topoisomerase IV. DNA gyrase and topoisomerases are responsible for negative supercoiling of DNA—an essential conformation for DNA replication in the intact cell. Inhibition of the activity of DNA gyrase and topoisomerase IV is lethal to bacterial cells.
Supercoiling- Basic concept
In duplex DNA, the two strands are wound about each other once every 10 bp, that is, once every turn of the helix. Double-stranded circular DNA can form either negative supercoils when the strands are underwound or positive supercoils when they are overwound. Negative supercoiling introduces a torsional stress that promotes unwinding or separation of the right-handed B-DNA double helix, while positive super coiling over winds such a helix- Figure-1
Figure-1- Negative and positive supercoils in DNA
Most naturally occurring DNA molecules are negatively supercoiled.
Negative supercoiling prepares DNA for processes requiring separation of the DNA strands, such as replication or transcription. Positive supercoiling condenses DNA as effectively, but it makes strand separation more difficult. Supercoiling markedly alters the overall form of DNA
The Supercoiling is controlled by a remarkable group of enzymes known as topoisomerases.
There are two classes of topoisomerases. These enzymes act by catalyzing a three-step process: (1) the cleavage of one or both strands of DNA, (2) the passage of a segment of DNA through this break, and (3) the resealing of the DNA break.
a) Type I topoisomerases relax DNA from negative supercoils by creating transient single-strand breaks in DNA without any expense of ATP. The type I enzymes have been further subdivided into type IA and type IB subfamilies based on their reaction mechanism (Figure-2)
Type I topoisomerases which form covalent linkages to the 5’ end of the DNA break are members of the type IA subfamily and type I enzymes which form covalent linkages to the 3’ end of DNA break are members of the type IB subfamily. With the backbone of one strand cleaved, the DNA can now rotate around the remaining strand, driven by the release of the energy stored because of the supercoiling. The rotation of the DNA unwinds supercoils (Figure-2).The enzyme controls the rotation so that the unwinding is not rapid.
Figure-2- Mechanism of action of two types of topoisomerases, Type I and Type II
b) Type II topoisomerases change DNA topology by making transient double-strand breaks in DNA and require ATP consumption.
c) A topoisomerase III has been described which makes transient single strand DNA breaks and is therefore a type I enzyme, and
d) A topoisomerase IV has been described which makes transient double strand DNA breaks and is therefore a member of the type II enzyme family.
All type II topoisomerases require ATP while Type I topoisomerases do not.
1. E. coli DNA Topoisomerase I
The enzyme has a preference for binding at single strand regions of DNA .This explains why the enzyme readily relaxes negatively, but not positively supercoiled DNA, as single stranded areas tend to be present in negatively supercoiled (underwound) DNA but not in positively supercoiled DNA.
2. E. coli DNA Topoisomerase III
Topo III is a type I enzyme and is therefore only capable of making transient DNA single strand breaks.
3 E. coli DNA Gyrase and DNA Topoisomerase IV
The type II topoisomerases are represented in E. coli by DNA gyrase and topoisomerase IV (topo IV). Unlike the type I topoisomerases, the type II enzymes all require ATP for catalysis.
DNA gyrase was discovered prior to topo IV .As a type II enzyme, it requires ATP. Gyrase is unique among topoisomerases because it is able to supercoil DNA. This supercoiling reaction is unique to prokaryotes. No active supercoiling activity has yet been described in higher organisms.
Topoiso-merase IV is a potent decatenate and plays a critical role in decatenating DNA after replication to allow proper partitioning of daughter chromosomes.
During DNA replication, type II topoisomerase, or topo II, plays an important role in the fork progression by continuous removal of the excessive positive supercoils that stem from the unwinding of the DNA strands. Topo II has the ability to cut both strands of a double-stranded DNA molecule, pass another portion of the duplex through the cut, and reseal the cut in a process that uses ATP. Hydrolysis of ATP by topo IIs inherent ATPase activity powers the conformational changes that are critical for the enzyme’s operation. Based on the DNA substrate, topo II can change a positive supercoil into a negative supercoil or increase the number of negative supercoils by two.
During transcription, positive supercoils accumulate in front of the transcription machinery, which can be removed by gyrase, and negative supercoils accumulate behind. It may be that a major function of E. coli topo I is to relax underwound DNA which accumulates behind an active RNA polymerase.
Drugs targeting the prokaryotic type II topoisomerases
DNA gyrase and topoisomerase IV are the molecular targets of two distinct groups of antibiotics; the coumarins (novobiocin) and the quinolones (ciprofloxacin). These two classes of drugs work by different mechanisms and are analogous to the mechanism of action of the two broad classes of anticancer drugs that target the eukaryotic enzymes. In general, topoisomerase targeting drugs as antibiotics or anticancer drugs either, inhibit the catalytic activity of the enzyme, or impair the ability of the enzyme to religate DNA after cleavage. Although drugs of the latter type necessarily inhibit catalysis as well, it is thought that the inhibition of DNA religation after cleavage leaves a cell with drug stabilized DNA breaks which are eventually converted into chromosomal breaks which result in cell death.
Mechanism of action of Ciprofloxacin
Representative of the second class of topoisomerase targeting drugs are the quinolones. These drugs interact with gyrase or topoisomerase IV and their DNA substrates and prevent the topoisomerase from relegating DNA after cleavage .This leaves the cell with double strand DNA breaks. If these breaks are not repaired, they interact with other intracellular proteins and become converted into irreversible double strand breaks that lead to cell death.
Mechanism of action of Novobiocin
These drugs inhibit the ATPase activity of gyrase. Because these coumarin antibiotics bind to a site that overlaps that of ATP they are competitive inhibitors.
These two gyrase inhibitors are widely used to treat urinary tract and other infections.
As regards other options-
Bacterial dihydrofolate reductase reduces dihydro folate to tetra hydro folate. Bacterial peptidyl transferase is involved in the process or protein synthesis. Bacterial RNA polymerase is required for the process of transcription, whereas DNA polymerase is required for the process of replication.
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