DNA Supercoiling: 7 Complete Quick Facts


Contents

What is Supercoiling? | DNA Supercoiling

The DNA of a cell as we all know is very compacted, inferring a higher level of structural organization. The folding mechanism of DNA packs the cellular DNA so that the information inside the DNA remains accessible.

We need to examine the basic structure of DNA carefully better to understand DNA replication and transcriptional process. We need to have prior knowledge about a crucial property of DNA structure that is supercoiling.

Supercoiling implies the further coiling of a coiled structure. Say, for example, a cord of a telephone is normally a coiled wire.  The wire between the body of the telephone and the receiver frequently incorporates at least one supercoils. The two DNA strands wrap around each other to produce the DNA double-helical structure. The coiling in the axis of DNA causes supercoiling. Supercoiling in DNA for the most part, is a sign of underlying structural strain. When there is no resultant DNA axial bending, the DNA is considered in relaxed state.

DNA supercoiling
Figure: DNA Supercoiling, levels of supercoiling found in DNA. Briefly, it resembles with the coiling of telephone wire
https://commons.wikimedia.org/wiki/File:Epigenetic_mechanisms.png

We may have anticipated that the process of compaction of DNA included some supercoiling. Maybe less unsurprising is that replication and transcription of DNA additionally influence and are influenced by supercoiling. The replication and transcription both require detachment of DNA strands and helical unwinding of DNA.

  • That DNA would itself twist and become supercoiled in compactly packed DNA in the cell would appear sensible, at that point, and maybe even insignificant.
  • However, several circular DNA molecules remain exceptionally supercoiled even after their extraction and purification.
  • This suggests, supercoiling is intrinsic property of DNA .It happens in every cell DNA and is exceptionally regulated by every cell.
  • Various measurable properties of supercoiling have been standardized, and the investigation of supercoiling has given numerous insights into the structure of DNA and its function.
  • This investigation is based on the ideas from topology and the investigation of properties of an object that doesn’t change under dynamic conditions.

For DNA, consistent deformations incorporate conformational changes because of thermal motion or protein interaction or other chemical agents; intermittent deformations include DNA strand breakage. For a circular DNA topological properties are not affected by structural changes in the DNA strands if strand breaks are not present. Topological properties are disturbed exclusively by sugar-phosphate backbone breaking and re-joining of either of the DNA strands. We currently examine the fundamental characteristics and physical basis of supercoiling.

Most Cellular DNA Is Underwound

To recognize supercoiling, we should initially concentrate on the characteristics of small circular DNAs like small viral DNAs and plasmids. If DNA does not have breaks in any of the strand, it is considered a closed circular DNA. Suppose the DNA of a circular DNA molecule aligns closely to form a B-form structure, as mentioned by Watson-Crick. The one turn of B-DNA consists of 10.5 bp; then, the DNA is relaxed instead of supercoiled.

Supercoiling is observed when DNA is subjected to structural strain. The purified circular DNA is very rarely found in the relaxed state, irrespective of its origin. Besides, DNA has a characteristic degree of supercoiling depending upon its source or origin. DNA structure is strained so that it can undergo supercoiling. In almost every event, the strain is an outcome of underwinding of the circular double-helical DNA.

  • That is to say, and the DNA has fewer helical turns than the B-DNA structure.
  • The B-DNA contains 10.5 base pairs (bp) in one turn.
  • So, an 84 bp fragment of a relaxed circular DNA would have eight helical turns. Or one turn for each 10.5 bp.
  • If one of these turns are removed, there would be (84 bp)/7 = 12.0 bp per turn, instead of 10.5 found in B-DNA.

It’s a deviation from the most stable form of DNA structure, and the DNA molecule is thermodynamically stressed accordingly. Usually, a lot of strain would be lodged by coiling the DNA axis on itself to frame a supercoil a portion of the strain in this 84 bp section would just get scattered in the untwisted structure of the bigger DNA particle).

On a basic level, strain can be induced by segregating the two DNA strands for a distance of about ten bp.

In circular DNA, strain presented by underwinding is generally lodged by supercoiling instead of strand segregation, Since coiling in the axis of DNA usually needs less energy than breaking the hydrogen bonds between complementary base pairs. 

Important Note: notwithstanding that the underwinding of DNA in vivo makes it simpler to segregate DNA strands, offering access to the genetic information they contain.

Each cell constantly underwinds its DNA with the help of enzymatic cycles, and the subsequent strained state constitutes a kind of stored energy. Cells keep DNA in underwound form so that it can easily undergo coiling. The underwinding of DNA is critically important for DNA metabolizing enzymes that need strand separation as a component of their function.

The underwound state is kept as such only in the case of closed circular DNA, or it must be bound with proteins (histones) so that strands would not wrap around one another. If strand of a circular DNA breaks which are segregated and non-protein bound, the free movement or rotation at the point will lead the underwound DNA to revert to its relaxed state immediately. In circular DNA, the number of helical turns imparts the degree of supercoiling.

Topological Linking Number defines DNA Underwinding

The topology gives various thoughts that are helpful to this conversation, especially the idea of linking number. Linking number is a property based on topological characteristics of double helical DNA since it doesn’t change if the DNA is deformed or bends without breaking. The linking number (Lk) is demonstrated in.

We should start by picturing the segregation of the two strands of a double helical circular DNA. If the two strands are connected, they are productively linked through a topological bond. Irrespective of all base stacking interactions and hydrogen bonds, the topological bond still joins the two strands.

If we consider one strands of a circular DNA as the surface (for example, a soap film), linking number is portrayed as the number of surface penetrations caused by the second strand. For a circular DNA, the linking number is always an integer value.

Conventionally, the linking number is positive when the strands of DNA double helix interwound in a right-handed helix. If the strands of DNA double helix interwounds in a left-handed helix, the linking number comes out to be negative. The negative linking numbers are not encountered in DNA practically.

When the circular DNA molecule is relaxed, the linking number can be easily determined by several base pairs in the DNA molecule by base pair per turn (~10.5). For a circular DNA having 2100 bp, it will exhibit a linking number of 200.

The linking number is calculated for those DNA molecules that do not break in either of the DNA strands. In the case of strand breakage, topological bonds do not exist; hence linking number remains undefined.

We are now be able to portray DNA underwinding as a result of linking number change. The connecting number in relaxed DNA, Lk0, is utilized as a source of perspective (reference). For a DNA molecule having a linking number = 200 (Lk0 = 200), if two turns are taken out or removed from this DNA molecule, Lk = 198. The equation can portray the change:

∆Lk = Lk – Lk0

∆Lk = 198 – 200 = -2

It is much better to express the change in linking number in terms of specific linking difference (σ) or superhelical density (this quantity does not depend upon DNA length). The specific linking number is mathematically defined as the number of turns removed (change in linking number) divided by several turns present in the relaxed state of DNA.

σ = ∆Lk/Lk0

so, for a DNA molecule having a linking number of 200, if two turns are removed from it, the specific linking difference becomes

σ = -2/200 = -0.01 

it demonstrates that 2 out of 200 (1%) of the total helical turns present in the B-DNA molecule are removed.

Positive Supercoiling | Negative Supercoiling

The degree of underwinding of cellular DNA is generally 5% to 7%; that is, -0.05 to -0.07. This negative sign shows that a change in linking number is expected to underwind or because of underwinding of DNA. The supercoiling triggered by underwinding is in this way characterized as negative supercoiling. Alternately, under certain conditions, DNA can be overwound, bringing about positive supercoiling.

Important note: The path of twisting in the DNA axis caused by positive supercoiling (overwound DNA) is the mirror image of twisting the DNA axis caused by negative supercoiling (underwound DNA).

Supercoiling is certainly not an arbitrary interaction; the supercoiling is generally directed by the torsional strain conferred upon the DNA by increasing or decreasing the linking number of the B-DNA. 

Linking number changes by one by the breakage of one DNA strand, turning one of the ends by 360o about the strand (unbroken) and rejoining the B-DNA’s broken ends.

This change does not disturb the number of base pairs or atoms in the circular DNA. Two types of circular DNA that vary in a topological property (for example, linking number) are called topoisomers.

Figure: Representation of Positive and Negative supercoiling in the DNA https://upload.wikimedia.org/wikipedia/commons/9/98/Subhash_nucleoid_06.png

Linking number can be divided into two primary components known as Twist (Tw) and Writhe (Wr). These are harder to portray than linking number. Yet, writhe might be considered an act of the coiling of the helical axis and Twist is determined as the spatial relationship of the neighbouring base pairs and local twisting. At this point, change in linking number results into a change in the proportion of strain that is normally made up for by writhe (supercoiling) and few by variations in the Twist, it is followed by the equation:

Lk Tw Wr

Tw and Wr are not necessarily integers. Writhe and Twist are geometrical properties instead of topological properties since distortions might change them in the circular DNA.

They cause supercoiling and making strand segregation much simpler. The underwinding of DNA works in conjugation with several other structural changes in the circular DNA. These are of lesser physiological significance however help show the impacts of underwinding.

A cruciform usually contains several unpaired bases; DNA underwinding assists with keeping up the necessary strand separation. The right-handed double helical DNA underwinding results into the formation of short patches of left-handed Z-DNA on places with the consistent nucleotide sequence with the Z-DNA.

Topoisomerase | Change in Linking Number | Introduction of Negative Supercoiling

DNA supercoiling is a process that impacts DNA metabolism. Each cell has specific enzymes with the sole capacity of underwinding or potentially relaxing the DNA double helix. The enzymes that cause a decrease or increase in DNA underwinding are known as topoisomerases; they usually change the linking number of DNA to maintain the supercoiling.

Figure: Mechanism of Action and Inhibition of Topoisomerase
https://commons.wikimedia.org/wiki/File:Topoisomerase_DNA.gif#/media/File:Topoisomerase_DNA.gif

These enzymes exhibit a particularly significant role in the process of DNA packaging and replication of DNA. Broadly topoisomerase is classified into two major types:

  • Type I topoisomerases breaks one strand from the double helical DNA. It passes the unbroken DNA strand through the gap and ligates the broken end; type I topoisomerase brings about a change in Lk by the increment of 1. 
  • Type II topoisomerases break both the strands of double helical DNA double helix and change Lk by the increment of 2.

The impacts of topoisomerases on the topology of DNA can be described by gel electrophoresis (agarose). A populace of similar plasmid DNAs with the equal linking number travels as a discontinuous band during electrophoresis. Topoisomers varying by as little as 1 Lk value can be isolated by this technique, so the change in linking number instigated by topoisomerases can be easily identified.

E. coli has four distinctive individual topoisomerases (I to IV). The type I (includes topoisomerases I and III): 

usually relax DNA double helix by eliminating negative supercoils (thus, increasing Lk value). How bacterial type I topoisomerases increase the linking number is explained in. A type II bacterial topoisomerase or DNA gyrase, is capable of inducing negative supercoils and eventually decreasing the Lk value.

It utilizes the energy of ATP to achieve this. To change the linking number of DNA double helix, type II topoisomerases separate the two strands of DNA double helix, and later it allows crossing another duplex through the break. The level of supercoiling of bacterial DNA is completely under topoisomerases I and II regulation. Eukaryotes additionally have topoisomerases both type I and type II.

The topoisomerases I and III belong to the class of type I enzymes; while the type II enzymes includes topoisomerases II-α and II-β. The eukaryotic type II topoisomerases can’t underwind DNA (induce negative supercoiling), yet they can relax negative and positive supercoiling.

Figure: Induction of Negative Supercoiling in DNA https://commons.wikimedia.org/wiki/File:Subhash_nucleoid_04.png#/media/File:Subhash_nucleoid_04.png

We will consider one likely the beginning of negative supercoils in eukaryotic cells in our conversation of chromatin in our later discussion.

DNA Compaction needs Supercoiling

Supercoiled DNA double helix is uniform in various regards. The Supercoiling is right-handed in a DNA molecule that is negatively supercoiled, and they tend to be narrow and extended instead of exhibiting a compact structure with numerous branches. At this superhelical density that is typically experienced in cells, length of the axis of supercoiling with branches, constitutes approximately 40% of the total DNA length.

This type of Supercoiling is known as plectonemic Supercoiling (it’s a combination of Greek words “plektos” means”curved,” and “nema” means “string”).

This term applies to any design with strands interlaced regularly and, and it is a decent description of the overall structure of supercoiled DNA in Plectonemic Supercoiling. The structure shown in separated DNAs in the lab doesn’t deliver adequate compaction to packed cellular DNA. The second type of Supercoiling, known as solenoidal, can be produced by an underwound DNA.

Figure: An example of Solenoidal Supercoiling Found in DNA https://en.wikipedia.org/wiki/File:Solenoid_30_nm_fibre_structure_closer_and_farther_away.png

Unlike plectonemic form (characterized by extended right-handed Supercoiling), solenoidal Supercoiling is characterized by tight left-handed turns. Irrespective that their structures are drastically different from each other, solenoidal and plectonemic are two types of negative Supercoiling that a similar fragment of underwound DNA can take up.

The two structures are frequently interconvertible. However, the plectonemic structure is more stable. But the solenoidal structure is often stabilized by protein binding, which is the structure found in chromatin. It gives a much higher level of compaction. In the Solenoidal Supercoiling, underwinding contributes toward DNA compaction.

Conclusions

In this article we had an in depth discussion about the mechanism of supercoiling of DNA and the enzymes and proteins involved in supercoiling process. We will continue to discuss more aspects of DNA in out upcoming posts. To know about the composition of DNA click here

Interview Q&A

Q1 Why is positive Supercoiling important?

Answer: Positive DNA supercoiling facilitates the unwinding of DNA from the histones and alters nucleosome structure in vitro; conversely, nucleosomes quickly form negative DNA supercoiled. Thus, it is considered that in every event of transcription, the positive Supercoiling is pushed after the RNA polymerase. The accumulated positive torsional stress triggers changes in nucleosomes and makes conditions in which polymerase proficiently transcribes through the entire nucleosomal array.

Q2 Is Supercoiling good or bad?

Answer: The Supercoiling of DNA is important since DNA is a very long molecule, and it has to fit into a cell with a radius of the order of micrometres. It requires repeated loop within loops of winding to fit, this type of winding is known as Supercoiling. It also regulates the transcription and replication process. 

Q3 How does positive Supercoiling of DNA increases?

Answer: Positive Supercoiling of DNA happens when the right-handed, double helix of DNA is bent much tighter (contorted in a right-gave design) until the helix starts to knot.

Q4 Negative Supercoiling in bacteria

Answer: Bacterial DNA generally exhibits negative Supercoiling in bacterial cells since it contains a deficiency of helical turns. In its B-DNA structure, the strands of the DNA double helix make one complete turn after every 10.5 base pairs. Increasing the number of turns makes the DNA double helix tighter and results in positing writhing because of the axial coiling in the DNA double helix. Eliminating turns through underwinding the duplex has the opposite impact, making the duplex writhe negatively.

Q5 Why is negative Supercoiling important?

Answer: Negative Supercoiling has a significant natural function of strand segregation of DNA during replication and transcription. Strand separation decreases the torsional stress in negative supercoiled DNA. In this way, it is energetically less expensive to segregate strands in negative supercoiled DNA than in the relaxed DNA, and the difference in energy is furnished by DNA relaxation.

Q6 Why does positively supercoiling DNA make DNA more stable in high temperatures?

Answer: Positive DNA supercoiling increases the number of links among two DNA strands. A conformation that gives resistance against heat-induced denaturation to the DNA double helix makes it stable under hyper-thermophilic conditions. the plasmids isolated from the bacterium living under hyper-thermophilic conditions exhibited a higher linking number as compared to the plasmids isolated from bacterium not living under these conditions.

Q7 Why does supercoil plasmid DNA migrate through agarose with the least resistance compared to the other form ex linear?

Answer: Because of its supercoiled nature, DNA fragments become smaller in size and thus experience less frictional obstruction from the gel—this results in the movement of this form of DNA being quicker than other forms.

Q8 What is the difference between constrained and unconstrained DNA supercoiling?

Answer: The word “unconstrained” is utilized to portray Supercoiling in “free” DNA because of topological strain. It isn’t supported or reliant upon proteins, for what it’s worth in eukaryotes, where roughly 147 base pairs of DNA are curled around the nucleosome, which is essentially a histone octamer made of 2 units histones H2A, H2B, H3 and H4. This type of Supercoiling is classified as “constrained” because the nucleosome structure constrains it.

Q9 Which enzyme helps cut one strand of DNA duplex to release the coiling of two strands?

Answer: Topoisomerase makes cut in strand and release coiling. Type I topoisomerases breaks one strand from the double helical DNA. It passes the unbroken DNA strand through the gap and ligates the broken end; type I topoisomerase brings about a change in Lk by the increment of 1. Type II topoisomerases break both the strands of double helical DNA double helix and change Lk by the increment of 2.

Q10 Are bacterial plasmids round, or are they supercoiled?

Answer: In many species, bacterial DNA is circular and negatively supercoiled. A plasmid purified from Escherichia coli in the mid-growth (exponential) phase is supercoiled to the degree of σ = −0.06. Inside the cell, the unconstrained Supercoiling is about half of this value.

Dr. Abdullah Arsalan

I am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology. Let's connect through LinkedIn(https://www.linkedin.com/in/abdullah-arsalan-a97a0a88/) or Google scholar(https://scholar.google.co.in/citations?user=AeZVWO4AAAAJ&hl=en).

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