The study of topology is the analysis of the geometrical properties and spatial relations of DNA, while these properties remain unaffected by any elastic deformation the molecule undergoes. This blog will offer a limelight on DNA supercoiling, the amount of twist on the DNA molecule, which determines the amount of strain on it. I have recently taken great interest in this topic, and thought it a great idea to share my takings from it with you all!
It is necessary to understand the different structural features of DNA before delving into the details of supercoiling of the molecule. There are various forms of DNA double helices, including B-form DNA, A-form DNA, and Z-form DNA. B-form is the most common of the three and makes up the high majority of DNA in cells - it is the classic Watson-Crick model that all biologists know of. Secondly, the A-form is similar to the B-form but is broader, less twisted (i.e. less bp/turn), and the bases are tilted and lie off the axis. It is currently unknown if this form occurs in vivo, but this has been proposed by numerous scientists. Finally, Z-form DNA is highly different from the previous two - it can be distinguished by the zig-zag path of its sugar-phosphate backbone, as well as lacking a major groove and having a deep and narrow minor groove. It is highly controversial whether this forms in nature, however, there are many hypotheses that it arises during certain cellular processes such as transcription and gene activation. Z-form DNA was actually the first DNA molecule to be analysed by X-ray crystallography, where the resulting structures came as a great surprise to the analysing scientists, as the helices were, in fact, left-handed, in comparison to the right-handed B-form helix.
As well as these varying forms of DNA duplexes, DNA triplexes and quadruplexes can form in DNA molecules! DNA triplexes occur when a DNA duplex associates with another DNA single strand to form a triple-stranded structure. One form of this, H-DNA, is formed when a polypyrimidine strand dissociates from a regular double helix, and lies in the major groove of another double helix strand, making the ‘single’ strand pair with the purine bases. These molecules have been given a lot of attention recently due to their therapeutic potential, as the third strand can be used to target a stretch of DNA in a sequence-specific manner. Repeating G's in the single-stranded section of telomeres can associate into tetrads, which can form parallel or antiparallel DNA quadruplexes! Furthermore, DNA structures such as Cruciforms can form in the presence of inverted repeats in double-stranded DNA, where a sequence is followed immediately (or soon after) by the same sequence in the opposite orientation. These types of sequences are known to occur in certain plasmids, and could therefore theoretically form cruciforms, however, this is unknown, as this formation is thermodynamically unstable due to the presence of unpaired bases. Another structure, a Holliday junction, is a four-armed intermediate that forms during genetic recombination. This occurs when two homologous DNA double helices are aligned, the cleavage of one strand of each helix occurs, base pairing is established with the intact strand of the other helix, and the strands are resealed.
The helix axis of DNA can also bend via the ‘Intrinsic curvature of DNA’, which is the bending of the helix axis that is the preferred conformation of a particular DNA sequence. Significant examples of this can be demonstrated by gel electrophoresis, where curved DNA molecules show reduced electrophoretic mobilities when compared with the sieving straight same-sized molecules. Furthermore, permuted DNA fragments (same sequenced molecules that start and finish at different positions) led to differences in electrophoresis mobility, although they had the same molecular weight. When A-tracts were found closer to the centre of a DNA fragment, mobility was at a minimum, however when close to the end mobility was at a maximum. A-tracts are short runs of adenine bases periodically repeated with a spacing of around 10-11 base pairs, which is also the helical repeat of B-DNA. There are many different proposed models as to why this occurs, however one has not yet been proven to discount the others.
In this way, it is clear that the DNA molecule is not as simple as GCSE and A-level textbooks make it out to be - there is a lot more to uncover than ‘two polynucleotide chains that coil around each other to form a double helix’! Although it can be thought of as a rigid, uniform structure, there is great structural diversity in DNA.
Now onto the main theme of this blog: DNA supercoiling. A possible problem occurs when a double helix is pulled apart by DNA helicase in replication, as when strands are pulled apart, the twisting of the DNA will tighten ahead of the separating strands, and the DNA will coil upon itself. If the whole DNA cannot rotate to relieve the stress that builds up, this will stop any further separation of the strands. This coiling or tangling of the DNA is the process of supercoiling, the inevitable result of trying to manipulate the DNA structure. The solution to this is that one or both DNA strands are broken and rejoined ahead of the replication fork to allow local rotation to occur at the breakpoint: this is carried out by enzymes called DNA topoisomerases. This phenomenon appeared unknowingly when research groups were investigating DNA molecules from the DNA tumour virus, polyoma. DNA from this virus fractioned into two components, I and II, during sedimentation analysis, which separates molecules according to size and compactness. These two circular DNA models, I and II, now known as supercoiled and nicked, were only distinguished by the breakage of one backbone phosphodiester bond and had several different properties, including I being more compact and unusually resistant to denaturation on heating or exposed to high pH. The solution for this came from electron micrographs, where images showed that there were many crossings of the DNA double strands in component I whereas component II comprised mainly of open rings. This suggested the I form to be a twisted circular form, with relative twisting of opposite ends prior to the 'theoretical closing' of the molecule. This represents the coiling of the DNA helix upon itself, the meaning of the term supercoiling. This supercoiling is essentially locked into the system, where the strain of this supercoiling cannot be released without breaking either one or two strands. As mentioned previously, although DNA may be geometrically contorted in many ways, this will not affect the supercoiling of the molecule. These findings led to a whole new field of studies, as behind this idea there are many ramifications, both theoretical and practical.
It is extremely important to be able to mathematically define supercoiling, so that DNA molecules differing in supercoiling, topoisomers, can be compared. The Linking number of a molecule is associated with the number of double helical turns in the original linear molecule. DNA has an inherent number of turns (discounting supercoiling), which can be found by dividing the length of the DNA in base pairs by the number of base pairs per turn of the helix (around 10.5 in B-DNA). This derived the formula Lk = N/h. It must be taken into account that this is a measure of a fundamental property of a closed-circular DNA molecule and has no meaning until both strands are sealed. Furthermore, when looking at describing geometrical confirmations, we have seen that a change in linking number leads to the coiling of the DNA upon itself, where these behaviours are complementary to one another. Twist describes how the individual strands of DNA coil around each other, and Writhe is a measure of the coiling of the helix axis in space. The Twist and Writhe are known to sum to the Linking number, and therefore any change in the linking number corresponds to a change in the twist and/or a change in the writhe.
DNA supercoiling is not the same in all conditions however! The two main studied conditions that have an effect on this topological property are the availability of positively charged ions in a solution and changes in temperature. Repulsion between negatively charged phosphate groups naturally unwinds the DNA helix. Therefore increasing the concentration of cations in a solution decreases the effective negative charge of these phosphates, therefore tightening the helix. Increasing temperature also has an effect on the helical repeat of DNA, as it results in more thermal motion of the molecule's components, thus leading to the gradual unwinding of the helix. In this way, a DNA molecule which is relaxed (lower levels of supercoiling) by a topoisomerase may not be relaxed when run on an agarose gel, due to potential differences in conditions.
So, why is this all important? It is an unavoidable issue that when looking into any part of the DNA molecule for any particular region, some form of DNA topology will need to be considered. The amount of a strand’s supercoiling affects a number of biological processes, such as compacting DNA and regulating access to the genetic code which strongly affects DNA metabolism and gene expression. DNA supercoiling has a direct influence on DNA-associated processes in vivo, mostly involving the interaction of specific proteins with the DNA, notably histone proteins. Therefore, given that topology has such a major impact on DNA, objectively the most important molecule on Earth, it is important for scientists to understand these processes in great detail!
Throughout my research going into this blog, I mainly focused on a book kindly given to me by my teacher. The name of this book is 'DNA Topology' by Andrew D. Bates and Anthony Maxwell. I hope this blog was of interest to you all!
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