Knitting and Knotting of DNA: The Cell's Ultimate Organizing Hack

 

How far is Pluto from Earth? It is an unfathomable distance to the human mind. Imagine a kite flying from your spool leaving the Earth’s atmosphere, crossing Mars, Jupiter, Saturn, Uranus, Neptune, finally reaching Pluto and then making it back to the spool in your hands, stretching over 16 billion kilometres. All of the DNA together from the 37 trillion cells of the human body could make a spool that makes that trip not once but six times. A single human cell holds 6 feet of DNA compressed into 6 micrometers in the nucleus. In conceivable perspective, it is similar to a thread that stretches from London to New York compressed into a tennis ball. Therefore, one must wonder if knots and tangles form in DNA when you compress and disturb it for replication and transcription, and how they would get resolved.

The long string of DNA wraps around histones to form nucleosomes, which are folded into chromatin fibers and self-interacting chromatin domains (TADs → Loops) with aid of cohesins, CTCFs, and other factors. This inherent wrapping and loop extrusion of DNA creates a precise and stable arrangement that prevents formation of knots and tangles. 

While the wrapping and folding is precise, it is not final. The DNA still has to unwind, rewind and move in the 3-Dimensional space for replication, transcription, and regulation. The DNA is negatively supercoiled, i.e., underwound in the cell, this is another inherent feature that allows for easy unwinding of DNA at controlled rates for polymerase access, thus hampering knots and tangles to an extent.

Nevertheless, disruption still occurs. Valdés et al. were the first to prove DNA knots exist in eukaryotic chromatin. They observed knots in yeast circular minichromosomes using high resolution 2D electrophoresis. Moving Replication forks and RNA polymerases cause torsional stress and supercoiling in genomic DNA i.e., disturb the  perfectly coiled, and folded loops. A special class of enzymes, DNA Topoisomerases, are the official detanglers that resolve this stress in the nucleus. 

Topoisomerases protect the fundamental process of the cell, such as, relax supercoiling ahead of replication forks, decatenate intertwined daughter DNA duplexes post replication, and prevent formation of R-loops during transcription. Since 1972, when DNA twisting was first seen, seven topoisomerases have been discovered in humans (TOP1, TOP1mt, TOP2α, TOP2β, TOP3α, TOP3β and Spo11). Topoisomerases create either reversible single strand breaks (type I topoisomerases) or Double Strand Breaks (DSBs) (type II topoisomerases). The tyrosine residues in the active sites of these enzymes attack the phosphodiester backbone forming phosphate-tyrosine linkages and creating reversible breaks. TOP1 forms these on the 3’ phosphate while TOP2 on the 5’. The attacks lead to covalently bound enzyme-DNA complexes known as topoisomerase cleavage complexes (TOPcc, TOP1cc, or TOP2cc). While TOP1 allows free rotation of DNA ends, reducing supercoiling. TOP2 creates a DSB, thus also creating a passage for a second DNA duplex to pass through and simplify DNA topology, i.e., removing knots.

However, as much as topoisomerases are saviours of the inevitable tangles in genomic DNA, they can also be dangerous when dysregulated forming irreversible DNA-protein crosslinks (TOP-DPCs). In fact, these enzymes, being absolutely essential for the fundamental processes for a cell’s survival, can likely form the largest proportion of toxic DNA adducts when deregulated or inhibited. The imbalance of topoisomerases can also create DSBs. For example, TOP1’s failure to relax supercoiling during transcription is linked to the formation of R-Loops (three stranded DNA-RNA assemblies formed when nascent mRNA pairs with template strand of DNA), the non-template ssDNA that is extruded in turn forms G4s (4-stranded DNA complexes, that can form with 4,3,2,or 1 strand when it folds). R-loops and G4s in turn form TOP2-mediated DSBs. However, the cell has also evolved to combat these damages with an array of DNA Damage repair proteins. 

Unlike normal DNA damage in a strand, the TOP-DPCs alter the phosphate backbone of DNA and create a large proteaceous shell around the phosphotyrosine linkage, blocking their access. Therefore different pathways are used to resolve these crosslinks, such as, conformational change induced by protein binding, proteolysis of bound topoisomerase, and nucleolytic cleavage of the flanking regions of DNA adjacent to the DNA.

Although, it is a well known fact that “prevention is better than cure.” Apart from the inherent arrangement and negative supercoiling of DNA, other preventive measures include, single strand binding proteins (SSBs) that bind to ssDNA during replication, RNA that binds to DNA opposite G4s and form G-Loops to prevent the knots from tightening. 

However, Valdés et al. state that a residual amount of steady-state fraction of DNA knots are produced by TOP2. These knots are independent of replication or transcriptive activities. They also observe that, during transcription these knots are significantly reduced, also owing to TOP2. The paradoxical role of TOP2 suggests that its activity blindly depends on the architecture of the chromatin fibres at that particular moment. Thus, despite the many mechanisms and systems put in place, Valdés et al. conclude that these knots are in fact, inevitable.

REFERENCES

1. Valdés A, Segura J, Dyson S, Martínez-García B, Roca J. DNA knots occur in intracellular chromatin. Nucleic Acids Res. 2018 Jan 25;46(2):650-660. doi: 10.1093/nar/gkx1137. PMID: 29149297; PMCID: PMC5778459.

2. Wojtaszek JL, Williams RS. From the TOP: Formation, recognition and resolution of topoisomerase DNA protein crosslinks. DNA Repair (Amst). 2024 Oct;142:103751. doi: 10.1016/j.dnarep.2024.103751. Epub 2024 Aug 16. PMID: 39180935; PMCID: PMC11404304.

3. Liu Z, Deibler RW, Chan HS, Zechiedrich L. The why and how of DNA unlinking. Nucleic Acids Res. 2009 Feb;37(3):661-71. doi: 10.1093/nar/gkp041. PMID: 19240147; PMCID: PMC2647305.

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        Cover image: https://www.iflscience.com/knot-as-strong-as-you-think-humans-are-bad-at-working-out-which-knots-are-strongest-77162

1. https://www.nature.com/scitable/topicpage/eukaryotic-genome-complexity-437/

2. https://www.nature.com/articles/s41580-022-00452-3