Biochemistry, DNA Structure

Issues of Concern

The primary issue of concern regarding the DNA structure is variations and mutations in DNA structure as proteins encoded by the mutated DNA generally have altered structure and function, adversely impacting the survival of the cell or organism. Mutations in DNA structure can take many forms, such as large or small insertions or deletions of base pairs or inversions and insertions of whole DNA segments between or within chromosomes.[4] In addition, several disorders are due to defects in cellular mechanisms associated with DNA, including replication, DNA repair, and transcription.[5][6][5][7]

Cellular Level

One significant difference between prokaryotes' and eukaryotes' DNA structure is that prokaryotic DNA molecules are circular and thus do not have free 5' and 3' ends. Circular DNA molecules are also found in eukaryotic mitochondrial and chloroplast DNA, evidence that supports the endosymbiotic theory of eukaryotic evolution.[8] In contrast, the ends of eukaryotic DNA molecules do not connect and are thus "free." Prokaryotes typically have one main circular chromosome, while eukaryotes have multiple linear chromosomes of varying sizes. For the specific purpose of decreasing their DNA size to ensure fitting inside a cell, prokaryotes employ DNA supercoiling.[9]

However, because eukaryotes have much more DNA than prokaryotes (3234 mega-base pairs vs. 4.4 mega-base pairs), they need to utilize a more complex strategy to position their DNA, which, if stretched from end to end, would be two meters long, properly inside a microscopic cellular space.[10] Specifically, this is done by sequential levels of coiling, starting with DNA wrapping around histone proteins forming a structure known as a nucleosome, then nucleosomes coiling to form chromatin fibers, and then chromatin further condensing into densely packed chromosomes.[11]

Molecular Level

A molecule of DNA is made up of two long polynucleotide chains consisting of subunits known as nucleotides. A nucleotide comprises a nitrogenous base, a pentose sugar, and at least one phosphate group (Figure 1a).[12] In the case of DNA, the sugar is 2’-deoxyribose, and thus it has no hydroxyl group attached to its 2’ (pronounced “two prime”) carbon; this is in contrast to ribose sugar in RNA, which does not have the 2’ position of its pentose sugar to be reduced (or deoxygenated). A phosphate group covalently binds to the 5’ carbon of 2’-deoxyribose. Since the 2’-deoxyribose and the phosphate group are always present, the nitrogenous bases they incorporate distinguish the four DNA nucleotides.[13]

A nucleotide can incorporate four main nitrogenous bases, two of which are purines and two that are pyrimidines (Figure 1b). Both purines and pyrimidines are heterocyclic aromatic compounds, as they contain nitrogen atoms in their carbon-based ring, which are essential for the hydrogen bonding that holds the two strands of the DNA molecule together. However, while pyrimidines are six-membered rings, purines consist of a five-membered ring fused to a six-membered ring. The two pyrimidines found in DNA are thymine (T) and cytosine (C), while the two purines are Adenine (A) and Guanine (G). The purines and pyrimidines differ slightly in structure, but their functional groups are attached to the same basic heterocyclic form. These nitrogenous bases are covalently bonded via a nitrogen atom to the 1’ carbon of the deoxyribose sugar in a nucleotide (Figure 1a).[1]

Although four major nitrogenous bases make up the nucleotides of DNA, other uncommon non-primary or modified bases have been found to exist in nature.[14] The most common modified bases in bacterial genomes are 5-methylcytosine, N6-methyladenine, and N4-methylcytosine. These modifications have been shown to protect DNA from restriction enzymes, which cleave DNA at specific sites. In all eukaryotic genomes, the most common modified base is 5-methylcytosine which is critical in regulating gene expression.[15]

Each strand of DNA is made up of a string of nucleotide subunits linked at their sugar moieties (Figure 2a). Specifically, nucleotides in a DNA strand are bound together via ester bonds between the phosphate group attached to their 5’ carbon and the hydroxyl group on the 3’ carbon of an adjacent nucleotide. This bond is known as a phosphodiester bond, and it forms via a condensation reaction during DNA synthesis. As a result, each strand of a DNA molecule has a series of nucleotides with their 5’ phosphate and 3’ hydroxyl group participating in phosphodiester bonds. Each strand of a eukaryotic DNA molecule has a “free” 5’ phosphate group on one end, not bonded to a hydroxyl group, and a “free” 3’ hydroxyl group on the other end, not bonded to a phosphate group. This asymmetry has led to the adoption of the convention where DNA is read in a particular direction, from its 5’ end to its 3’ end. The sequence of nucleotides that make up a molecule of DNA is referred to as its primary structure.[16]

A DNA molecule consists of two chains of polymerized nucleotides running side-by-side, joined together by hydrogen bonds forming between their nitrogenous bases (Figure 2a). Notably, the nucleotides bond in a particular fashion, with A pairing with T and G pairing with C; A and T pairing is by two hydrogen bonds, and C and G by three. These specific pairings result in about a 1 to 1 ratio of pyrimidines and purines in any given cell, a concept known as Chargaff’s rule. This pairing scheme is called complementary base-pairing and is the most energetically favorable pairing possible. DNA is structured so that the sugars of each strand are on the outside, while the bases hydrogen bond on the inside, resulting in what is known as the sugar-phosphate backbone. Thus, two chains of sugar-phosphate backbones run side-by-side with complementary paired nitrogenous bases hydrogen bonding between them. Notably, the two strands of a DNA molecule run in an antiparallel fashion so that the 5’ end of one strand is the 3’ end of the other.[17] This base pairing of nucleotides between the two strands of a single DNA molecule is called DNA’s secondary structure.

The three-dimensional shape of a DNA molecule, or its tertiary structure, is a right-handed double helix (Figure 2b). The hydrogen-bonded bases on each strand are stacked in parallel and run perpendicular to the sugar-phosphate backbone. As indicated by its x-ray diffraction pattern, the bases are regularly spaced at 0.34 nm apart along the axis of the helix.[18] Additionally, there are about ten pairs of bases per turn, as a complete turn of the helix is made every 3.4 nm. DNA has a +36-degree rotation per base pair (bp) and a helical diameter of 1.9 nm.[18] When focusing on the backbone of the DNA helix, two helical grooves exist with different widths, known as the minor and major grooves (Figure 2b). The minor groove describes the space between the two antiparallel DNA strands that run closest together, while the major groove describes the space where they are furthest apart. These specific dimensions describe the B form of DNA, the major form present in most stretches of DNA in a cell.[19] This is in contrast to DNA’s much rarer A and Z forms. The A form is a right-handed double helix with less distance between the bases (0.256 nm), and thus more bases per turn (11 bp per turn) and a smaller helical rotation per base pair (+33 degrees).[19][20] Z DNA is a left-handed double helix and is most present in the human genome, where many purines and pyrimidines are alternating in succession (i.e., in a sequence such as GCGCGCGCGCG).[20] DNA primarily takes the B form, in contrast to any other form, because it is the most energetically stable tertiary structure.[20][21]

A notable property of DNA is the ease of reversible separation of its two strands due to hydrogen bonds being relatively weak compared to covalent bonds. This is important because fundamental cellular processes such as DNA replication and RNA transcription rely on proteins accessing individually separated strands of DNA. Thus, during these processes, proteins known as helicases move down the DNA molecule and unwind the two strands by disrupting the hydrogen bonding between bases. However, when the cellular processes requiring strand separation are complete, the complementary strands can easily re-anneal. This property of reversible separation can be experimentally induced via the heating and cooling of a DNA molecule and is referred to as denaturation or “melting.”[22][23]

One notable structural phenomenon of DNA tertiary structure is supercoiling, or the coiling of the larger, already coiled DNA molecule. Specifically, in a DNA molecule that has its ends fixed, such as in the circular DNA found in prokaryotes or the smaller DNA segments that make up a larger chromosome in eukaryotes, separation of the individual strands of DNA during cellular processes causes the DNA to twist-up past the points of strand separation, leading to strain on the larger DNA structure.[9] This transient over-winding of the larger DNA structure when separating individual strands is known as positive supercoiling (Figure 5). Every cell has enzymes that keep DNA actively underwound to compensate for this, resulting in perpetual negative supercoiling, where the larger DNA structure coils in a left-handed fashion. This results in the strands of DNA needing less energy to be separated and keeps the molecule primed for easy separation in the events of transcription and DNA replication. 

Function

The unique structure of DNA is ultimately responsible for its function as being the material that stores and transmits genetic information from one generation to the next. Specifically, the four nitrogenous bases that comprise the sequence of nucleotides in a DNA molecule enable an enormous amount of information stored in minimal space. DNA’s sugar-phosphate backbone and helical structure make it more stable, less prone to damage, and more compact; however, the hydrogen bonds that hold the strands of DNA together make it more accessible for its biological functions as they are individually weak but cumulatively strong. Also, the complementary base pairing of nucleotides in DNA enables accurate semiconservative replication as each strand carries identical genetic information and serves as an independent template during DNA replication.[13][24]

Clinical Significance

DNA mutations have a fundamental role in the pathophysiology of multiple conditions ranging from congenital and developmental diseases to cancer.[25][26] One important example is sickle-cell anemia, an inherited genetic disease that predominates in individuals of African descent. This disease is a direct result of a single point mutation of an A to a T in the gene that encodes beta-globin, resulting in the sixth amino acid in beta-globin’s polypeptide chain changing from glutamic acid to valine.[27] Consequently, an individual homozygous for this mutation will have hemoglobin with mutated beta-globin subunits, known as HbS, that aggregate into crystalline arrays when deoxygenated. This mutation in hemoglobin results in the deformation of erythrocytes into a sickle-like shape, making them prone to block capillaries, leading to hemolytic anemia, episodes of vascular occlusion, and reduced blood flow.[27][28]