Size matters – the importance of DNA packaging – GENAWIF – Society for Natural Compound- and Drug Research

Summary

The following article introduces the importance of DNA size management due to limited cellular space, as it is demonstrated with the DNA and cell size of the Escherichia coli bacterium. The very principles of DNA packaging are discussed using a simple sticks-and-strings model that you can try at home. Since DNA packaging is a much more sophisticated process with various structural proteins and enzymes involved, the bacterial enzymes DNA gyrase and DNA topoisomerase I are introduced as well as an easy and well-established assay to monitor their DNA modifying activity. The assay can be used to screen for new DNA gyrase inhibitors, an important target of medical antibiotics.

Section 1 – DNA is used to store high amounts of biological information

All the information about the cellular structure of an organism, its proteins needed for biochemical reactions, and genetic circuits to adapt to changing environmental conditions are stored physically on DNA molecules. Simply spoken, every biological detail of an organism is encoded in its DNA, which is quite much of information. Thus, as we will see, the absolute size of DNA exceeds the actual cell size, which is why the DNA has to be packaged and condensed to fit in its host cell.

Section 2 – The DNA structure and terms that are used to refer to DNA length

Firstly, we need to explain the units and terms that are used to express the length or size of DNA molecules. DNA molecules are made up of two strands wound around each other to make the familiar double helix one sees in illustrations (Figure 1). One strand is made up of a chain of bases joined along a sugar-phosphate backbone, and each base in a strand pairs with a base in the opposite strand giving a unit called a base pair (bp)in the DNA double helix ¹. Thus, the length of a DNA molecule can be expressed as a number of base pairs (bp).

Figure 1: A simplified scheme showing the structure of DNA. DNA helices consists of two single molecular strands that are attached to each other via base paring with hydrogen bonds, represented here by vertical lines between the bases adenine (A) and thymine (T) and cytosine (C) und guanine (G). The bases are covalently bound to a sugar-phosphate backbone, and their serial sequence represents genetic information which can be read and interpreted by cells. Due to the arrangements and the connection of the different DNA components to each other, the natural conformation of DNA is helical.

DNA molecules are classified depending on their size, structure and function. For example, chromosomes are the largest DNA molecules in a cell with all the genes that are necessary for fundamental cellular function. In higher organisms the chromosomal DNA is the sum of several different chromosomes. In contrast, the DNA in bacteria that makes up the bacterial chromosome is, in most cases, a single long DNA molecule joined together at the ends to make a circle ².

Section 3 – Escherichia coli´s DNA- and cell size

The chromosome of Escherichia coli K12, a common laboratory bacterial strain, has a size of 4.6 mega bp which is 4.6 million bp of DNA ³.Knowing that each base pair is approximately 0.34 nm long ⁴, the physical length of the “DNA string” of the E. coli genome can be calculated to have a length of 0.34 nm x 4,600,000 bp = 1,564,000 nm, which is a DNA molecule about 1.5 mm long, a dimension visible to the human eye (Figure 2). If a 1.5 mm string is used to form a perfect circle which would be a closer fit to the real DNA structure of the E. coli chromosome, the diameter of this circle can be calculated to approximately 0.5 mm, a size also still visible by the naked eye.

Figure 2: The length of the Escherichia coli genome compared to its actual size within the bacterial cell. The DNA is composed of a string of covalently bound nucleotides. Each nucleotide has one of four different bases, which form hydrogen bonds with corresponding complementary bases of another string, resulting in two parallel strings of DNA. Given the length of 0.34 nm between base pairs (bp), the E. coli genome with a size of 4.6 million base pairs is 1,564,000 nm long, which equals 1,564 µm or 1.5 mm. Based on this length, the diameter of a perfect circular molecule which would be closer to the real structure of the E. coli genome can be calculated to 0.5 mm (500 µm). Thus, the chromosomal DNA of E. coli is still more than 100 to 200 times bigger than its actual cell size.

Since E. coli cells are so small that we cannot see them with the naked eye, 0.002-0.004 mm long and 0.001 mm in diameter ^5–7, it is clear that the chromosomal DNA has to be packed into a smaller form, which scientists call the nucleoid ⁸ (Figure 2).

Section 4 – A simplified model of DNA packaging

When being packaged, the DNA double strand crosses over itself several times forming super coils, which is the form of condensed or packed DNA within the cell. In this condensed form, the DNA can be stored very compactly, like folding a 2-meter-long ruler to fit in your pocket.

A simple model that can be reproduced at home with two strings and two sticks can nicely demonstrate the principles of DNA packaging (Figure 3). The two strings represent two parallel DNA single strands. If each end of this double strand is tied to a stick forming two parallel strands, and if you turn one stick around while holding the other in position, and fold the DNA to bring the sticks closer together, the DNA becomes twisted. As both sticks to come closer upon repeated folding, the DNA curls up and forms coils which reduces the tension overall in the molecule.

Figure 3: A simple model of DNA packaging to fit the long DNA molecule neatly into a small space. The linear DNA consists of two strands of DNA strings, connected by the hydrogen bonds between complementary DNA bases (vertical lines between the horizontal strings). If both ends of the linear DNA string are tied to fixed ends, and one end is twisted, the DNA becomes twisted as well. Twisted DNA is neatly folded, and sticking to this model, a tension is built up between both ends which is released by allowing both ends to come closer to each other, while the DNA relaxes by forming coiled structures.

As with everything in Biology, the packaging (condensation) and un-packaging (de-condensation) of DNA is quite complicated and involves several enzymes and structural proteins that modify and stabilize the shape of the DNA. Enzymes that actively participate in DNA condensation and de-condensation belong to the so-called topoisomerase protein family (nicely reviewed for example by Champoux ⁹).

DNA packaging is not a once in a lifetime event, because each time a gene is transcribed or the DNA is replicated, the DNA must be locally or totally de-condensed, respectively. In principle, one can compare DNA with a book in which a specific page is opened (de-condensed) for copying (transcribing) a certain passage of genetic information. The page is then turned over, closing (condensing) the passage, and the transcript of this passage is then used to perform an assignment in the cell. DNA condensation and de-condensation are, thus, essential for cellular function and cell replication, and impairing enzymes responsible for this process is generally lethal, which is why bacteria-specific topoisomerases like DNA gyrase are valuable targets for antibiotic treatment ¹⁰.

Section 5 – Visualizing the activity of DNA packaging enzymes via simple agarose gel electrophoresis

Using DNA gyrase and topoisomerase I, enzymatically assisted packaging and un-packaging of DNA can nowadays be simply demonstrated in the laboratory. But first of all, what does these enzymes do? In a nutshell, DNA gyrase actively increases the number of twists, and the higher the number of twists, the more looped and compact the DNA gets. In more detail, DNA gyrase grasps two strands. Then, one strand is cut and the other strand is pulled through the generated opening, followed by rejoining of the cut strand by DNA gyrase ¹⁰. On the other hand, DNA topoisomerase I actively relaxes the supercoiled DNA by cutting and re-joining the strands after allowing unwinding to occur ⁹ (Figure 4).

Now that we know our enzymes, we need to think about the DNA sample that can be used to be enzymatically modified and investigated. So far, we discussed the size and packaging of DNA using genomic DNA as an example. However, for experimental purposes, Plasmid DNA is a better choice. Plasmids are circular but smaller, autonomously replicating DNA molecules that co-exist with the chromosomal DNA in the bacterial cell, often in high copy number. Since small plasmid DNA is much easier to isolate and to handle than chromosomal DNA, plasmid DNA is a good experimental material to use to investigate DNA Gyrase and Topoisomerase I activity (Figure 4).

Figure 4: A method to investigate DNA topoisomerase activity. Circular relaxed plasmid DNA can be converted to supercoiled DNA via DNA gyrase, and supercoiled DNA can be converted back to relaxed plasmid DNA by DNA topoisomerase I. The action of both enzymes can be monitored if the DNA is analysed after enzymatic treatment. Although the relaxed- and supercoiled DNA still have the same absolute size, the supercoiled DNA is much more compact and can run through the meshwork of an agarose gel faster within a certain time than the less compact relaxed DNA on the same gel.

Lastly, we need to analyse the structural changes made by the enzymes to our DNA substrate. DNA conformation analysis is quite simple using an agarose gel, which is a polymer building a meshwork serving as a molecular sieve with a pore size depending on the agarose concentration used. The denser this meshwork is the more time is needed for DNA molecules to squeeze through when pulled along by an electrical current (electrophoresis).

The gel is put in a chamber with conducting buffer, the DNA samples are then loaded in pockets within the gel, and by applying an electrical field via two electrodes in the chamber, dissolved substances with an electrical charge wander through the gel towards the oppositely charged electrode. With the negatively charged cathode at the top, the negatively charged DNA molecules are pulled through the agarose gel towards the positively charged anode at the bottom. The more compact the DNA molecules are, the faster they can move through the agarose gel meshwork. Thus, packaged DNA with less steric hindrance would run further through the gel compared to un-packed DNA of the same number of bp within a certain time.

This kind of assay can easily be used to screen for additional DNA gyrase inhibitors that could be new antibiotic candidates. Although already having antibiotics at hand that inhibit DNA gyrase activity, antibiotic usage is often accompanied by antibiotic resistance development ¹¹. Thus, it is always reasonable to look for alternative antibiotics.

For example, in another study, the herein explained gel electrophoresis assay was used to monitor DNA gyrase activity after treatment with allicin from garlic ¹². We also summarized this study in another article on our website, entitled “Allicin from garlic inhibits the essential bacterial enzyme DNA gyrase, a common target for medical antibiotics”, which would be the perfect follow-up for you to read!

Jan Borlinghaus, 13.09.2022

References

Alberts, B.; Johnson, A.; Lewi, J.; Raff, M.; Roberts, K.; Walter, P. The Structure and Function of DNA. In Molecular Biology of the Cell.; Garland Science: New York, 2022.
diCenzo, G. C.; Finan, T. M. The Divided Bacterial Genome: Structure, Function, and Evolution. Microbiology and Molecular Biology Reviews 2017, 81 (3), e00019-17. https://doi.org/10.1128/MMBR.00019-17.
Blattner, F. R.; Plunkett, G.; Bloch, C. A.; Perna, N. T.; Burland, V.; Riley, M.; Collado-Vides, J.; Glasner, J. D.; Rode, C. K.; Mayhew, G. F.; Gregor, J.; Davis, N. W.; Kirkpatrick, H. A.; Goeden, M. A.; Rose, D. J.; Mau, B.; Shao, Y. The Complete Genome Sequence of Escherichia Coli K-12. Science 1997, 277 (5331), 1453. https://doi.org/10.1126/science.277.5331.1453.
Langridge, R.; Wilson, H. R.; Hooper, C. W.; Wilkins, M. H. F.; Hamilton, L. D. The Molecular Configuration of Deoxyribonucleic Acid: I. X-Ray Diffraction Study of a Crystalline Form of the Lithium Salt. Journal of Molecular Biology 1960, 2 (1), 19-IN11. https://doi.org/10.1016/S0022-2836(60)80004-6.
Volkmer, B.; Heinemann, M. Condition-Dependent Cell Volume and Concentration of Escherichia Coli to Facilitate Data Conversion for Systems Biology Modeling. PLoS One 2011, 6 (7), e23126–e23126. https://doi.org/10.1371/journal.pone.0023126.
Reshes, G.; Vanounou, S.; Fishov, I.; Feingold, M. Timing the Start of Division in E. Coli: A Single-Cell Study. Phys Biol 2008, 5 (4), 046001. https://doi.org/10.1088/1478-3975/5/4/046001.
Pierucci, O. Dimensions of Escherichia Coli at Various Growth Rates: Model for Envelope Growth. J Bacteriol 1978, 135 (2), 559–574. https://doi.org/10.1128/jb.135.2.559-574.1978.
Verma, S. C.; Qian, Z.; Adhya, S. L. Architecture of the Escherichia Coli Nucleoid. PLOS Genetics 2019, 15 (12), e1008456. https://doi.org/10.1371/journal.pgen.1008456.
Champoux, J. J. DNA TOPOISOMERASES: Structure, Function, and Mechanism. Annu. Rev. Biochem. 2001, 0 (errata). https://doi.org/10.1146/annurev.bi.70.010101.200002.
Collin, F.; Karkare, S.; Maxwell, A. Exploiting Bacterial DNA Gyrase as a Drug Target: Current State and Perspectives. Applied Microbiology and Biotechnology 2011, 92 (3), 479–497. https://doi.org/10.1007/s00253-011-3557-z.
MacLean, R. C.; San Millan, A. The Evolution of Antibiotic Resistance. Science 2019, 365 (6458), 1082. https://doi.org/10.1126/science.aax3879.
Reiter, J.; Hübbers, A. M.; Albrecht, F.; Leichert, L. I. O.; Slusarenko, A. J. Allicin, a Natural Antimicrobial Defence Substance from Garlic, Inhibits DNA Gyrase Activity in Bacteria. International Journal of Medical Microbiology 2020, 310 (1), 151359. https://doi.org/10.1016/j.ijmm.2019.151359.