B&B - DNA Topology

#Infodump4

DNA structure constitutes repeatedly intertwining 2 polynucleotide strands that are free ended. The free ends make untangling this linear DNA possible. However, in case of natural DNAs, the easy rotation (unwinding) as a consequence free ends is restricted.

Reasons:

Plasmids, bacterial chromosomes, mitochondrial DNA, chloroplast DNA and many viral DNAs are circular. Hence no ends for rotation.
The extreme length and entrainment in chromatin subjects eukaryotic DNA to restricted rotations of its ends.
The linear eukaryotic chromosomes also consist of regions of DNA that are attached to the nuclear matrix similarly DNA regions are attached to the membrane in case of viruses. These lead to formation of DNA loops that are closed ended like circular DNA.
Region of DNA between two proteins too have ends that are rotationally restricted.

These segments of DNA with rotationally constrained ends are referred to as Topological Domain.

What topology means?

Mathematical definition: “properties that are preserved through deformations, twistings, and stretchings of objects but not tearing or gluing”.

Thus, is case of DNA the topological properties are those that can be altered specifically by breaking either one or both strands of the double helix. These properties include DNA underwinding and overwinding, knotting, and tangling.

What is the importance?

Despite the constraints on rotation, two strands of the DNA must separate for DNA replication, transcription, recombination, and mitosis. Topology affects all these process that require the double helix to be opened. Therefore, understanding topology of DNA and how it is altered to enable the previously mentioned cellular processes is important.

To understand the major concepts, the topology of circular DNA has been considered. However, these concepts remain applicable to all cases.

Circular DNA

Membrane-DNA interaction

Protein-DNA interaction

Linking Number (Lk)

Lk: the number of times one DNA strand has to crosses the other in order to be entirely separated from each other.
Lk is always an integer and is an invariant topological property. In other words, the only way to change Lk is introducing breaks in one or both strands of DNA.
Mathematically,

Lk = Tw + Wr

Twist (Tw): the number of helical turns of one strand about the other.

Writhe (Wr): the spatial pass of the double helix axis. In other words, the number of times the double helix crosses itself.

Neither Tw nor Wr are only/always integers. Neither Tw nor Wr are topological invariants.

Writhe can positive or negative as depicted below.

"In all living systems DNA is negatively supercoiled"

What does this statement mean?

Basics:

The previously discussed (#Infodump3) B-form of DNA has 10.5 basepairs per helical turn. This form is said to be 'relaxed' DNA. The linking number of such DNA is symbolised as Lk°. Addition or removal of twists in circular DNA will alter the number of basepairs per turn and lead to contortion of the structure (example: '8' structure depicted above). Such contortions are referred to as 'supercoil'.
Mathematically,

ΔLk = Lk - Lk°

The extent of supercoiling is measured by difference between Lk and Lk°. This difference is called as Linking difference (ΔLk ).

ΔLk > 0 --> DNA is positively supercoiled

ΔLk < 0 --> DNA is negatively supercoiled

Since, ΔLk is length dependent, the supercoiling in molecules of different sizes is compared via calculating the term 'σ' i.e. superhelical density. σ is independent of length.

σ = ΔLk / Lk°

σ --> -ve --> underwound DNA

σ --> +ve --> overwound DNA

Converting Supercoiled DNA to Relaxed DNA requires introduction of nicks (breaking phosphodiester bond/s) in the strands. The nicks will allow free rotation of strands allowing the removal of writhe and ultimately the supercoiled DNA is now relaxed.

Answer:

In all living systems (prokaryotic and eukaryotic) ΔLk < 0, therefore, σ is negative i.e. DNA is globally underwound by ~6%.

WHY IS THIS IMPORTANT?

The underwound regions have tendency to partially unwind i.e. they make strand separation easily attainable. As discussed previously, strand separation in essential for cellular processes.

EXCEPTION : THERMOPHILES

Thermophiles have overwound DNA in order to avoid denaturation due to high temperatures at which these organisms thrive.
This means strand separation is more energy intensive in case of thermophiles than in other organisms.

As linking number can only be altered by creating nicks in DNA:

Topoisomerases

are enzymes that are responsible for introducing these single-stranded or double-stranded breaks. These enzymes are involved in decatenating, disentangling and unknotting DNA.

Topoisomerases are of 2 types:

Topoisomerase I : Alters the linking number in a single step, does not require ATP for its activity and are responsible for single strand breaks. Lk has single unit change.
Topoisomerase II : Alters the linking number in a two steps (depicted below), does require ATP/NADH for its activity and are responsible for double-stranded breaks. Lk changes by 2 units (the writhe is altered).

Diagram Explanation: Topoisomerase II cleaves the top duplex, passes a segment of the uncut duplex through the break and finally reseals the break.

DNA gyrase (type of Topoisomerase II) found in prokaryotes introduces negative supercoils instead of removing them. This enzyme is responsible for negative supercoiling of DNA in prokaryotes.

Reverse gyrase are found in thermophiles and are responsible for positive supercoiling.

Mechanism - Topoisomerase I:

Type 1 topoisomerase has tyrosine residue at its active site. The hydroxyl group of tyrosine covalently attaches to a DNA phosphate and thus creates a nick (breaks phosphodiester linkage) in one DNA strand. This break allows rotation of double helix in order to release the strain. ATP is not required as the energy source since the original phosphodiester bond energy is stored in the phosphotyrosine linkage. Hence, this reaction is reversible. Which means after the favourable rotation, the phosphodiester bond regenerates allowing the enzyme to dissociate.

Diagram explanation - Mechanism (topoisomerase II):

Step 1: A DNA duplex associates with the DNA binding in DNA-gate region. ATP binds and this aids in capturing of the second DNA duplex.

Step2: ATP binding also initiates dimerization of the ATPase domains leading to the closure of the ATPase domains i.e. the N-gate.

Step 3: This stimulates the cleavage of the first bound DNA duplex thus creates a DNA 'gate', hence, the region is also referred as DNA gate. This allows passing the second duplex through the break.

Step 4: The first duplex is religated and the complex dissociates from the DNA by expelling second duplex via C-gate.

As established previously, DNA gyrase is responsible for negative supercoiling of DNA in prokaryotes. What about eukaryotes?

Nucleosomes are responsible.

The next infodump will focus on DNA Packaging: Nucleosomes and Chromatin.

References:

James D. Watson, Tania A. Baker, Stephen P. Bell, Alexander Gann, Michael Levine, Richard Losick - Molecular Biology of the Gene (2013, Benjamin Cummings) .
Iwasa, J., Marshall, W. F., & Karp, G. (2015). Karps cell and molecular biology. Hoboken:Wiley.
Mirkin, S. M. (2001). DNA Topology: Fundamentals. Encyclopedia of Life Sciences, (c), 1–11. https://doi.org/10.1038/npg.els.0001038
Deweese, J. E., Osheroff, M. A., & Osheroff, N. (2008). DNA Topology and Topoisomerases: Teaching a "Knotty" Subject. Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 37(1), 2–10. https://doi.org/10.1002/bmb.20244
Ahmad, M., Xue, Y., Lee, S. K., Martindale, J. L., Shen, W., Li, W., Zou, S., Ciaramella, M., Debat, H., Nadal, M., Leng, F., Zhang, H., Wang, Q., Siaw, G. E., Niu, H., Pommier, Y., Gorospe, M., Hsieh, T. S., Tse-Dinh, Y. C., Xu, D., … Wang, W. (2016). RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals. Nucleic acids research, 44(13), 6335–6349. https://doi.org/10.1093/nar/gkw508
Lee, & Wang,. (2019). Roles of Topoisomerases in Heterochromatin, Aging, and Diseases. Genes. 10. 884. 10.3390/genes10110884.