Question

Supercoiled DNA is slightly unwound compared to relaxed DNA and this enables it to assume a more compact structure with enhanced physical stability. Describe the enzymes that control the number of supercoils present in the \(E .\) coli chromosome. How much would you have to reduce the linking number to increase the number of supercoils by five?

Short answer

Answer: The enzymes controlling the number of supercoils in the E. coli chromosome are topoisomerases, including type I topoisomerases (Topoisomerase I/TopA and Topoisomerase III/TopB) and type II topoisomerases (DNA gyrase/Topoisomerase II/GyrAB). To increase the number of supercoils by five, the linking number must be reduced by -5 for type I topoisomerases or by -6 for type II topoisomerases.

Step-by-step solution

Introduce topoisomerases

Topoisomerases are enzymes responsible for controlling the level of supercoiling in DNA, including the E. coli chromosome. These enzymes classify into two categories: type I topoisomerases and type II topoisomerases. Both types of topoisomerases work by breaking, unwinding/winding, and rejoining DNA strands to control the number of supercoils in the molecule.

Describe type I and type II topoisomerases

Type I topoisomerases temporarily cleave a single DNA strand and allow it to rotate around the remaining intact strand to change the linking number by ±1. E. coli has two type I topoisomerases: Topoisomerase I (also known as TopA) and Topoisomerase III (TopB). Type II topoisomerases temporarily cleave both DNA strands and pass another DNA helix through the break before resealing the strands. This process changes the linking number by ±2. In E. coli, the primary type II topoisomerase is DNA gyrase (also called Topoisomerase II or GyrAB), which introduces negative supercoils into DNA.

Introduce linking number and supercoiling

The linking number (Lk) is a topological property of the DNA molecule that describes the number of times one DNA strand wraps around the other strand in a double helix. Relaxed B-form DNA has 10.5 base pairs per helical turn, giving it a linking number of approximately 10.5 multiplied by the number of turns. Supercoiling occurs when DNA becomes overwound (positively supercoiled) or underwound (negatively supercoiled) compared to the relaxed state.

Calculate necessary reduction in linking number to increase supercoils by five

To increase the number of supercoils by five, we'll need to use the equation: Δsupercoils = ΔLk. Since we want to increase the number of supercoils by five, we're aiming for a change in supercoils of +5. The change in linking number (ΔLk) needed to achieve this depends on the action of the enzymes: - For type I topoisomerases, each action changes the linking number by ±1. Thus, to achieve an increase of +5 supercoils, we would need to reduce the linking number by -5 (ΔLk = -5). - For type II topoisomerases like DNA gyrase, each action changes the linking number by ±2. Thus, to achieve an increase of +5 supercoils, we would need to reduce the linking number by -6 (ΔLk = -6), as three actions of introducing negative supercoils (3 × -2) would result in an overall change of +5 supercoils. Note that we cannot achieve exactly +5 supercoils with type II topoisomerases acting alone. So to increase the number of supercoils by five, we would need to reduce the linking number by -5 if we're considering the action of type I topoisomerases, or reduce it by -6 with type II topoisomerases actions.

Key concepts

Topoisomerases

Topoisomerases play a crucial role in maintaining the structure and function of DNA by managing its supercoiled state. They are enzymes that alter the topological states of DNA, such as its coiling and tension, by creating transient breaks in the DNA strands. Without topoisomerases, vital cellular processes such as DNA replication and transcription would be hindered by the physical stress induced by supercoiled DNA.

In E. coli, topoisomerases like Topoisomerase I and III (type I) and DNA gyrase (type II) are essential for regulating supercoiling. Topoisomerase I and III make single-stranded cuts to alleviate supercoils one at a time, while DNA gyrase can introduce or remove supercoils in pairs by cutting both strands of the DNA helix. Understanding how these enzymes function sheds light on how cells preserve their genetic integrity during cellular processes.

Linking Number

The linking number (Lk) is a mathematical expression quantifying the entwined state of the two strands of a DNA molecule. It represents the total number of times one DNA strand wraps around the other in a closed circular DNA molecule. The concept is critical for understanding DNA supercoiling, which occurs when the DNA helix winds or unwinds from its relaxed state.

The number of base pairs per helical turn in a relaxed DNA molecule is usually 10.5. Any deviation from this can lead to a change in the linking number, which in turn affects DNA's supercoiling. An increase or decrease in supercoiling can be achieved by modifying the linking number, which is precisely what topoisomerases are designed to control. By manipulating the linking number, these enzymes can reduce the strain on the DNA and ensure its efficient function within the cell.

E. coli chromosome

The chromosome of Escherichia coli, a common bacterium used as a model organism in genetics, is a singular, closed circular molecule of DNA. Due to its closed-loop structure, the DNA must be compacted to fit within the bacterial cell, and this compaction results in supercoiling.

The E. coli chromosome typifies supercoiling's significance, as the bacterium's survival and replication are highly dependent on the proper organization and segregation of its chromosome. This intricacy is managed through the action of topoisomerases, which are vital for supporting the DNA's dynamic structure during the cell cycle, facilitating processes such as replication, transcription, and recombination.

DNA stability

DNA stability is pivotal for preserving the genetic information and avoiding mutations that can result in disease or cell death. Supercoiling enhances DNA stability by compacting the molecule and making it less susceptible to damage from external forces. However, too much supercoiling can also be detrimental, leading to DNA breaks or hindering essential processes such as replication and transcription.

Thus, the balance of supercoiling, managed by topoisomerases, is vital for DNA stability. Negative supercoiling introduced by enzymes like DNA gyrase can help in DNA unwinding, which is essential for replication and transcription, while positive supercoiling, typically found ahead of replication forks, helps to regulate the unwinding process. In summary, DNA stability hinges on the meticulous and dynamic interplay between supercoiling and the enzymatic actions that control it.