Definition for View source for DNA Structure and Replication Process and History

The discovery of DNA is argued to be one of the most important and significant scientific findings in the history of biology. While this statement may seem somewhat of an over-exaggeration, it is one of the founding models that have revolutionized the way we do science and the science that we do. Our knowledge of DNA has helped us explore fields of biology that were once limited to one’s imagination, this includes fields in microbiology, medicine, pathology, agriculture, and the environment. Hence, our knowledge of DNA has catalyzed the way we look at science and nearly all living things. 

Deoxyribonucleic acid, or DNA for short, is the blueprint of all life, giving instruction and function to organisms ranging from simple one-celled bacteria to complex eukaryotic organisms. It is the genetic material found in each cell of our body (excluding red-blood cells) that codes for how each cell behaves, interacts with neighbouring cells, how the cell looks like, and how it develops. DNA is passed on from generation to generation via reproduction in higher organisms or simply by cell division in prokaryotes. In eukaryotes, DNA is found in the nucleus, whereas in prokaryotes, DNA is found in the cytoplasm (recall that prokaryotes do not have membrane-bound organelles). 

DNA is a large polymer made up of a single, basic building block called a nucleotide. A nucleotide is a relatively complex molecule made up of a pentose sugar, nitrogenous base, and phosphate. The source of variation in DNA is found in the nitrogenous bases. In fact, four different nitrogenous bases exist in DNA, namely adenine (denoted: A), guanine (G), thymine (T), and cytosine (C). Adenine and guanine are considered purines because they possess a double-ring structure, while thymine and cytosine are considered pyrimidines because they have a single-ring structure. Essentially, purines consist of two pyrimidines rings fused together. Prior to the discovery of the structure of DNA, a scientist named Erwin Chargoff revealed that the amount of purines is always equal to the amount of pyrimidines when extracted from the [[nucleoplasm]] of various organisms. In addition, he revealed that the percentage of adenine is equal to that of thymine, and the proportion of guanine is equal to that of cytosine. Collectively, these discoveries were labelled as Chargoff’s rules. 

Since a nucleotide is made up of three separate components, these molecules somehow have to be held together by chemical bonds. The chemical bond that holds the sugar pentose to the nucleic acid is a glycosyl bond. The bond that holds the sugar to the phosphate is an ester bond. The five carbons that make up the sugar molecule are also labelled and their position plays a major role in the formation of the DNA polymer. The pentose sugar also has a specific name, deoxyribose. Unlike its counterpart, ribose, the sugar molecule, deoxyribose, lacks one less oxygen, hence the prefix deoxy-. 

To understand how this relatively simple molecule we have come to know as a nucleotide could account for the large number of characteristics in all species, it was necessary for scientists to understand how these nucleotides come together. All they really knew was that the nucleus contained (1) large amounts of nucleotides and (2) Chargaff’s rules. In order to create a DNA model, it was vital for scientists to visualize its actual structure in real-time. To do this, a scientist named Rosalind Franklin used X-ray diffraction analysis to determine its structure. X-ray diffraction is probably one of the most powerful tools that scientists use to visualize a molecule. Remember, molecules are simply too small to be seen. Molecules are things we can visualize and, in the case of X-ray diffraction, we visualize them by literarily taking a photo of the molecule of interest. 

In X-ray diffraction, a cube of crystallized molecules is bombarded with X-rays. These X-rays are deflected by the atoms in the molecule of interest, producing a pattern of lighter and darker lines on photographic film. The molecule’s 3-dimensional structure is then deduced using complex mathematical analysis. Using the X-ray detraction pattern discovered by Rosalind Franklin, it was found that DNA has three consistent numbers associated with its structure, namely, 2 nanometers (nm), 0.34 nm, and 3.4 nm. It also appeared to have a helical shape. Using Franklin’s information, in addition to Chargaff’s rules, James Watson and Francis Crick were the first scientists to build the infamous structure of DNA in 1953 - a double helix. In fact, this was such a prized accomplishment that Watson, Crick, and Maurice Wilkins (the scientist who provided Franklin with crystallized DNA) were awarded the Nobel Prize in Physiology/medicine in 1962 for their efforts. Since Rosalind died of cancer prior to 1962, she was not eligible for the prize because the Nobel Prize cannot be award to the deceased, thereby causing controversy in the scientific community. Moreover, prior to Watson and Crick’s discovery, there was a great deal of controversy as to the helical nature of DNA. Some scientists proposed a three-helical structure, while others proposed a one helical structure. 

According to Watson and Crick’s DNA model, adenine always hydrogen-bonds with thymine, while cytosine always hydrogen-bonds with guanine. Although hydrogen bonds are relatively weak, many hydrogen bonds taken together are quite strong, which explains DNA’s high stability as a polymer. This is consistent with both Chargaff’s proportionality rule and the 2 nm number that was constant in the X-ray diffraction pattern produced by Franklin because otherwise, if a purine, for instance, hydrogen bonded with another purine, it would be wider in some parts of the double helix and smaller in other parts. This is also the reason why pyrimidines cannot bind with pyrimidines. Collectively, this is known as complementary base pairing. An important consequence of the complementary base pairing is that if you know the sequence on one strand, you also know the sequence on the complementary strand. 

Moreover, according to their model, one complete turn of the helix occurs every ten nucleotides. Thus, if one turn is made every 3.4 nm and there are 10 nucleotides to a complete turn, each nucleotide is separated by a distance of 0.34 nm, since 3.4 nm / 0.34 nm = 10 nucleotides to a turn. Furthermore, DNA consists of two antiparallel strands of nucleotides. The nucleotides in one strand are bonded together and arranged above or below each other, perpendicular to the axis of the entire molecule. Since there are two strands running antiparallel to one another, this means that one strand goes upwards and the other strand goes downwards. In fact, one strand runs in the 5’ to 3’ direction, while the other strand runs in the 3’ to 5’ direction. Therefore, every DNA molecule has intrinsic directionality. Each nucleotide is held together by phosphodiester bonds between the phosphate of one nucleotide and the 3rd carbon of another. When phosphodiester bonds are formed, a condensation reaction occurs, where one molecule of water is lost in the process. The nucleotide all the way at the 3’ end does not have 

Immediately after the structure of DNA was discover by Watson and Crick, its structure immediately suggested how it was able to replicate. It was suggested that the hydrogen bonds between complementary bases could break, thereby allowing the DNA helix to unzip. Then, each single DNA strand could act as a template to build the complementary strand, resulting in two identical DNA molecules. 

In order to try to prove this hypothesis, two scientists named Matthew Meselson and Franklin Stahl started experimenting with bacteria in 1958. They began their study by culturing bacteria in a medium containing an isotope of nitrogen (N15). Since nitrogenous bases are composed of nitrogen, after several generations in this medium, the (N15) would get incorporated into the cultured bacteria’s DNA. The DNA from the bacteria was extracted and centrifuged in cesium chloride, a chemical that forms a density gradient after being centrifuged over a long period of time. A density gradient is a variation in density over an area. In cesium, the density is greatest at the bottom. When the DNA was centrifuged, it landed close to the bottom. Next, the same bacteria were cultured in N14 for several generations. If Watson and Crick’s hypothesis were true, only one band would be visible, a band containing DNA of both a mixture of N14 and N15. If two bands resulted – that is, one higher in the gradient and one at the same location of the previous test, it would suggest conservative replication – where both parental strands of DNA remain together following DNA replication. It was found that there was only one large band in the middle, that is, between the N14 band and the N15 band, indicating that this layer of intermediate DNA contained both N14 nucleotides and N15 nucleotides. This suggested that the double-stranded DNA is half conserved following the replication process such that the new double-stranded DNA contains one parental strand and one daughter strand – a process known as semi-conservative replication. This experiment is known as the Meselson-Stahl Experiment and has been called ‘the most beautiful experiment in biology’. 

Although Watson and Crick’s idea of breaking the hydrogen bonds is correct, simply unzipping DNA like you would with a zipper is inaccurate. In fact, DNA cannot fully unwind because of its large size compared with the size of the cell. For example, the diameter of a cell is approximately 5 µm. The length of DNA in a single human chromosome is approximately 1 cm, which is 2000 times larger than 5 µm. Unlike a zipper that have only one starting point, DNA replication begins in multiple areas at once, called origins of replication. When hydrogen bonds are broken at these origins of replication, replication bubbles all throughout the linear DNA, making replication can go n-times as fast. Bacteria (prokaryotes) on the other hand have circular DNA – as opposed to linear – and so they only have one origin of replication – this is fine considering the relatively small size of prokaryotic DNA. 

To expose a template strand, the two parent DNA strands must be unravelled and kept separate. The enzyme DNA helicase first unwinds the double helix by breaking the hydrogen bonds between the complementary base pairs holding the two DNA strands together. Since they are complementary, nucleotides have the propensity to anneal, so to prevent this from happening, two individual strands are kept apart by single stranded binding proteins (SSBPs). SSBPs bind to the exposed DNA single strands and block hydrogen bonding. The enzyme DNA gyrase relieves any tension brought about by the unwinding of the DNA strands during replication. As soon as these areas of single stranded DNA are exposed, complementary strands are built. 

As the two strands of DNA are disrupted, the junction where they are still joined is called the replication fork. DNA replication proceeds toward the direction of the replication fork. As mentioned before, in eukaryotes more than one replication fork may exist on a DNA molecule at once because of the multiple sites of origin, ensuring the rapid replication of DNA. In prokaryotes, DNA polymerase I, II, and III are the three enzymes known to function in replication and repair. In eukaryotes, five different types of DNA polymerase are at work. The enzyme that builds the complementary strand using the template strand as a guide in prokaryotes is DNA polymerase III. DNA polymerase III functions only under certain conditions. In order to polymerize the new strand, a free 3’-OH group is required to add a new nucleotide to the existing strand. However, before DNA polymerase III can polymerize a new strand of DNA, it needs a temporary primer that possesses a free OH. The enzyme primase lays down RNA primers that will be used by DNA polymerase III as a starting point to build the new complementary strands. Once the temporary RNA primer as been, DNA polymerase III adds the appropriate deoxyribonucleoside triphosphates to the free 3’ end of the primer, building a new strand in 5’ to 3’ direction on both template strands – this is known as the leading strand. The energy in the phosphate bonds is used to drive the process. The leading strand is built continuously toward the replication fork. The lagging strands – found on both template strands – are composed of short segments of DNA, known as Okazaki fragments. They are built discontinuously away from the replication fork. Understand that Okazaki fragments are also built by DNA polymerase III and require a primer to start building. 

As replication proceeds, DNA polymerase I excises the RNA primers and replaces them with the appropriate deoxyribonucleotides. DNA ligase joins the gaps in the Okazaki fragments and leading strand by the creation of a phosphodiester bond. Following this, DNA polymerase I and DNA polymerase III proofread by excising incorrectly paired nucleotides at the end of the complementary strand and adding the correct nucleotides.