Case study – Urinary Tract infection

Case Details

A 30-year-old female presents with burning on urination and blood in her urine. She is diagnosed with a urinary tract infection. She is prescribed an antibiotic that inhibits an enzyme that prevents extreme supercoiling of DNA. Super coiling of DNA results from unwinding at a replication fork. This enzyme breaks and rejoins DNA chains. Which of the following is the most characteristic of this enzyme?

A. Helicase

B. Single strand binding protein

C. Topo isomerase

D. DNA polymerase I

E. DNA polymerase III

Answer- The correct answer is C- Topoisomerase.

The Supercoiling is controlled by a remarkable group of enzymes known as topoisomerases.

Supercoiling

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 DNA can form either negative supercoils when the strands are underwound or positive supercoils when they are overwound (figure-1).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.

Supercoiling

Figure-1- Negative and positive supercoiling

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.

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.

There are two main classes of topoisomerases-

a) Type I topoisomerases relax DNA from negative supercoils by creating transient single-strand breaks in DNA without any expense of ATP.

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.

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.

The type II topoisomerases are represented in E. coli by DNA gyrase and topoisomerase IV (topo IV).

DNA gyrase and topoisomerase IV are the molecular targets of two distinct groups of antibiotics; the coumarins (novobiocin) and the Quinolones (ciprofloxacin).

The Quinolones, including Nalidixic acid and its fluorinated derivatives (ciprofloxacin, levofloxacin, and moxifloxacin), are synthetic compounds that inhibit the activity of the bacterial enzyme DNA gyrase as well as topoisomerase IV.

Inhibition of the activity of DNA gyrase and topoisomerase IV is lethal to bacterial cells. Humans have different topoisomerases than prokaryotes. The inhibitors of these topoisomerases are used as anticancer drugs.

Topoisomerase targeting drugs acting  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. 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.

As regards other options

During replication helicase separates the DNA strands by breaking hydrogen bonds between the two strands, the resultant torsional stress is relieved by topo isomerases (Figure-2).

Topoisomerase

Figure-2- DNA helicase unwinds, Topoisomerases relieve torsional stress and Single strand binding proteins stabilize the unwound DNA structure

 Single strand binding proteins bind the single DNA stands to prevent reannealing and to prevent the attack from nucleases.

DNA Polymerase I is involved in DNA repair and primer removal.

DNA Polymerase III is the  main enzyme for DNA replication.

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