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TEXT Components of DNA To understand the structure of DNA, it is important to understand the individual components of DNA. It is composed of pentose sugar, aromatic bases (a purine or pyrimidine ring) and phosphate groups. The many variations in the structures of the bases and sugars, and in the structural relationship of the bases to the sugar, give rise to differences in the helical structure of DNA. A. Pentose sugar Two kinds of pentoses are found in nucleic acids. The recurring deoxyribonucleotide units of DNA contain 2’deoxy-D-ribose or simply, deoxyribose (Fig. 1), which is the reason for the name deoxyribose nucleic acid. The ribonucleotide units of RNA contain D-ribose, hence ribose nucleic acid. In nucleotides, both types of pentoses are in the β- furanose form, i.e. in a closed five-member ring. The oxygen atom present at the second carbon of ribose is missing in deoxyribose, hence its name 2’deoxyribose. The position of carbon atoms of pentose sugars are denoted as 1’ , 2’ , 3’ , 4’ and 5’ in order to differentiate them from the corresponding positions in DNA bases which are not marked by a prime (‘). The sugar moiety of DNA is one of the most flexible and dynamic parts of the molecule. Fig. 2 shows structures of the common sugar conformations that are found in the various forms of DNA. The sugar ring is easy to envision if one thinks of an envelope. In the envelope form, the four carbons form a plane at the corners of the body of the envelope. The oxygen is at the position representing the top of the envelope flap. The oxygen can be bent out of the plane of the body of the envelope twisting the C2’ and C3’ carbons relative to the other atoms, resulting in various twist forms of the sugar ring. To form the C2’endo form of the ribose sugar, C2’ twists up from the plane of the four carbons. To form the C3’ endo, C3’ twists down out of the plane of the four carbons. B. Organic base The organic bases found in nucleic acids are heterocyclic compounds containing nitrogen in their rings; hence they are called nitrogenous bases. The bases found in nucleotides are substituted pyrimidines and purines. Unsubstituted pyrimidines and purines are not found in biological systems, but a number of substituted derivatives are present, which are classified as pyrimidines or purines, depending on the parent molecule from which they are derived. a. Pyrimidine bases: Pyrimidine bases consist of a six membered pyrimidine ring, which is similar to the benzene ring except that it contains nitrogen in place of carbon at positions 1 and 3 (Fig. 3). Therefore, pyrimidines contain four carbons and two nitrogen atoms. The carbons and nitrogens of the purine rings are numbered 1 to 9. DNA contain two major pyrimidines: i. Thymine: Thymine contains a methyl group at the C5 position with the carbonyl groups at C4 and C2 positions (Fig.3 ), so it is 5-methyl, 2,4-dioxo pyrimidine. ii. Cytosine: Cytosine contains a hydrogen atom at the C5 position and an amino group at C4 (Fig.3 ), so it is 2oxo-4-amino pyrimidine. It will be pertinent to mention that RNA contains uracil in place of thymine. Only rarely thymine occurs in RNA or uracil in DNA. In uracil, a keto group (=O) is present at the 2nd and 4th carbon, so it is 2,4-dioxo pyrimidine (Fig.3 ). The only difference between uracil and thymine is the absence of a methyl group at position C5 position. b. Purine bases Purine is a bicyclic structure consisting of pyrimidine fused to an five membered imidazole ring. The five member ring of purine has nitrogen in the place of carbon at positions 7 and 9 (Fig.4 ). Therefore, purines contain five carbons and four nitrogen atoms. The carbons and nitrogens of the pyrimidines are numbered 1 to 6. The two major purine bases present in DNA are: i. Adenine: In adenine, an amino group is present at position 6 (Fig.4 ), so it is 6-amino purine. ii. Guanine: In guanine, an amino group is attached with the 2nd carbon and a keto group is found at position 6 (Fig.4 ), therefore it is 2-amino-6-oxo purine. Each commonly occurring pyrimidine and purines can be drawn in two tautomeric forms. Adenine and cytosine can exist as either amino- or imino- forms, and guanine, thymine and uracil can exist as either lactum or lactim forms (Fig.5 ). The two forms for each base exist in equilibrium, but under the conditions found inside most cells, the amino and lactum tautomers are more stable, and therefore, predominate. The nitrogenous base is linked to position 1 on the pentose ring by a glycosidic bond (Fig.6 ). The purine bases are bonded at the 9 nitrogen, while the pyrimidines bond at the 1 nitrogen. This bond is said to be in the β (up) configuration with respect to the ribose sugar, in contrast to the α (down) position of the hydrogen. The base is free to rotate around the glycosidic bond. The two standard conformations of the base around the glycosidic bond is syn and anti. The anti conformation reflects the relative spatial orientation of the base and sugar as found in most conformations of DNA, for example, B-form DNA. The syn conformation is found (in conjugation with a different sugar pucker) in Z-form DNA. The purines and pyrimidines are well-suited to their roles as the informational molecules of the cell. The differential placement of hydrogen bond donor and acceptor groups gives the bases the unique structural identity that allows them to serve as the genetic information. The hydrogen atoms of amino groups provide hydrogen bond donors, where as the carbonyl oxygen and ring nitrogen provide hydrogen bond acceptors. All the carbon atoms of purine and pyrimidines are sp2 hybridized (that is none are saturated); thus they are planar. This flatness is important in the organization of bases within the helix, since it allows the bases to stack uniformly within the helix and this stacking helps to protect the chemical identity of the bases. C. Phosphate group Phosphoric acid has three reactive hydroxyl groups of which two are involved in forming the sugar phosphate backbone of DNA. Nucleoside and nucleotide A base linked to sugar is called nucleoside; when a phosphate group is added, the base-sugar-phosphate is called nucleotide. ATP and AMP are nucleotides, whereas the unphosphorylated form, adenosine, is a nucleoside. Fig.7 depicts the structures and names of the four major deoxyribonucleotides (deoxyribonucleoside 5’monophosphate). Nucleotides provide the building blocks from which nucleic acids are constructed. The nucleotides are linked together into a polynucleotide chain by a backbone consisting of an alternating series of sugar and phosphate residues. The 5’ position of one pentose ring is connected to the 3’ position of the next pentose ring via a phosphate group. So the sugar-phosphate backbone is said to consist of 5’-3’ phosphodiester linkages (Fig.8 ). Thus, the covalent backbones of nucleic acid consists of alternating phosphate and pentose residues, and the characteristic bases may be regarded as side groups joined to the backbone at regular intervals. It is noteworthy that DNA is hydrophilic. The hydroxyl groups of the sugar residues form hydrogen bonds with water. The phosphate groups in the polar backbone are completely ionized and negatively charged at pH 7, thus DNA is an acid. These negative charges are generally neutralized by ionic interactions with positive charges on proteins (proteins that contain an abundance of the basic residues arginine & lysine), metal ions (Mg2+) and polyamines. All the phosphodiester linkages in DNA strands have the same orientation giving each linear nucleic acid strand a specific polarity (Fig.9). The terminal nucleotide at one end of the chain has a free 5’ group and the terminal nucleotide at the other end has free 3’ group. It is conventional to write nucleic acid sequence in the 5’→3’ direction, that is, from the 5’ terminus at the left to the 3’ terminus at right. When DNA is broken into its constituent nucleotides, the cleavage may take place on either side of the phosphodiester bonds. Depending on the circumstances, nucleotides have their phosphate group attached to either the 5’ or the 3’ position of the pentose (Fig.10). Therefore, the two types of nucleotides released from nucleic acids are nucleoside-3’monophosphates and nucleoside-5’-monophosphates. All the nucleotides can exist in a form in which there is more than one phosphate group linked to the 5’ position. An example is shown in Fig.11). The bonds between the first (α) and second (β), and between the second (β) and third (γ), phosphate groups are energyrich and are used to provide an energy source for various cellular activities. The 5’ triphosphates are the precursors for nucleic acid synthesis (Fig.12). Nucleic acid synthesis occurs by a reaction in which the 5’ end of the incoming triphosphate reacts with a 3’-OH group at the end of the polynucleotide chain. A bond is formed from the α phosphate to the 3’-OH of the sugar at the end of the polynucleotide chain, and the two terminal phosphate groups (γ and β) of the triphosphate are released, in the form of a single molecule called pyrophosphate. The physical structure of DNA DNA is a duplex molecule, i.e. it consists of two chains arranged in anti-parallel manner with the nitrogenous bases facing each other. However, there are some examples of viral DNA which are single-stranded. It is sometimes useful to describe nucleic acid structure in terms of increase in levels of complexity like primary, secondary and tertiary. A. Primary structure The primary structure of a nucleic acid is its covalent structure and nucleotide sequence (Fig.13). It is in these sequences where the genetic information is stored and because the skeleton is the same for all, the difference in the information lies in the different sequence of nitrogenous bases. This sequence has a code, which determines an information or otherwise, as the order of the bases. B. Secondary structure Any regular, stable structure taken up by some or all of the nucleotides in a nucleic acid can be referred to as secondary structure. It is a double helix structure (Fig.14). Double helix model explain the storage of genetic information and the mechanism of DNA replication. It was postulated by Watson and Crick, based on X-ray diffraction and the equivalence of bases, whereby the sum of adenines and guanines is equal to the sum of thymines and cytokines. C. Tertiary structure Refers to how DNA is stored in a confined space to form the chromosomes. Varies, depending on whether the organism is prokaryote or eukaryote. In prokaryotes the DNA is folded like a super-helix, usually in circular shape and associated with a small amount of protein (Fig.15). The same happens in cellular organelles such as mitochondria and the chloroplasts. In eukaryotes, since the amount of DNA in each chromosome is very large, the packing is more complex and compact due to the presence of proteins such as histones and non-histones. Discovery of DNA structure: The deoxyribonucleotides and their ability to form polynucleotide were discovered by Levene in 1931. But Levene postulated that the four deoxyribonucleotides occurred in a regularly repeated tetra nucleotide sequence like AGCTAGCT…. and so on. A. Erwin Chargaff’s Ratios A most important clue to the structure of DNA came from Erwin Chargaff & his colleagues in 1949. The data collected by them from DNAs of many different species, led to the following conclusions: a. The base composition of DNA generally varies from one species to another (Table-1 ). b. DNA specimens isolated from different tissues of the same species have same base composition. c. The base composition of DNA in a given species does not change with the organisms age, nutritional state or changing environment. d. In all DNAs, regardless of the species, the number of adenine residue is equal to the number of thymine residues (A=T), and the number of guanine residues is equal to the number of cytosine residues (G=C). From these relationships it follows that the sum of purine residues equals the sum of pyrimidine residues; that is A+G=T+C. Table-1:Chargaff’s DNA database composition in various species: Species A% T% G% C% Homo sapiens 31.0 31.5 19.1 18.4 Drosophila melongaster 27.3 27.6 22.5 22.5 Zea mays 25.6 25.3 24.5 24.6S Neurospora crassa 23.0 23.3 27.1 26.6 Escherichia coli 24.6 24.3 25.5 25.6 Bacillus subtilis 28.4 29.0 21.0 21.6 These quantitative relationships, sometimes called “Chargaff’s rules”’ were a key to establishing the three dimensional structure of DNA and yielded clues to how genetic information is encoded in DNA and passed from one generation to the next. Therefore, tetranucleotide hypothesis of Levene proposing that DNA has repeating units of one of these four bases was disapproved. B. Francis Wilkin’s diffraction data Rosalind Franklin, working with Maurice H.F. Wilkins at King’s College (London, England) studied isolated fibres of DNA by using the X-ray diffraction technique, a procedure in which beam of parallel X-rays is directed on a regular, repeating array of atoms (Fig.16). The beam is diffracted (=broken up) by the atoms in a pattern that is characteristic of the atomic weight and the spatial arrangement of the molecules. The diffracted X-rays are recorded on a photographic plate. X-ray diffraction pattern revealed that DNA is a helical structure which had two distinctive regularities of 0.34 nm and 3.4 nm along the axis of the molecule. C. Watson & Crick’s double helix model Watson and Crick received the Nobel Prize in 1962 for their model of DNA. Using information generated by Chargaff and Franklin, Watson and Crick built a model of DNA. Their model was consistent with both Chargaff's rules and dimensions of DNA polymer provided by Franklin's photograph of X-ray diffraction of DNA. The double-helix model proposed by Watson and Crick in 1953 consists of two helical DNA chains coiled around the same axis to form a right-handed double helix (Fig.17). The hydrophilic backbones of alternating deoxyribose and negatively charged phosphate groups are on the outside of the double helix, facing the surrounding water. The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis of the helix. The spatial relationship between these strands creates a major or larger groove and minor or smaller groove between the two strands. In the Watson-Crick structure, the two strands of the helix are anti-parallel i.e., their 5’-3’ phosphodiester bonds run in opposite directions. Each base of one strand forms hydrogen bonds with a base of the other strand, forming a base pair. Only the lactum and amino tautomers of each base accommodate such hydrogen bonding. Guanine pairs with cytosine, and adenine with thymine. These base pairs maximize hydrogen bonding between potential sites. Accordingly, G/C base pairs have three hydrogen bonds and A/T base pairs have two. This feature of double-stranded DNA accounts for Chargaff’s earlier discovery that the ratio of A to T and G to C is 1:1 for a wide diversity of DNA molecules. Because A in one strand pairs with T in the other and G pairs with C, the strands are complementary and can serve as a template for each other. During replication, the two strands of a DNA molecule uncoil, and the unpaired bases in the single stranded region of the two strands bind with their complementary bases present in the cytoplasm as nucleotides. These nucleotides become joined by phosphodiester linkages generating complementary strands of the old ones. This provides for almost errorfree high fidelity replication of the genetic material. To account for the periodicities observed in the X-ray diffraction pattern, Watson and Crick used molecular models to show that the vertically stacked bases inside the double helix would be 0.34 nm apart and that the secondary repeat distance of about 3.4 nm could be accounted for by the presence of 10 nucleotide residues in each complete turn of the double helix. DNA double helix or duplex is held together by two sets of forces: a. Hydrogen bonding between complementary base pairs A hydrogen bond is a short, non-covalent, directional interaction between covalently bound H+ atom (donor) and a negatively charged acceptor atom. The acceptor is provided by electrons on a carbonyl oxygen [C=O] or the lone pair electrons on nitrogen [N:] (Fig.18). In the DNA double helix, the nitrogen and oxygen atoms involved in hydrogen bonding are separated by 0.282 0.292 nm. In A=T base pair, two hydrogen bonds are separated by 0.282nm and 0.291nm and in the G≡C base pair, three hydrogen bonds are separated by 0.284nm and 0.292 nm. In DNA, hydrogen bonds have 2-3 kcal/mol weaker than most hydrogen bonds (3-7 kcal/mol), which is due to geometric constraints within the double helix. b. Base-stacking interactions Since the aromatic bases are planar, they can stack nicely on one another. The stacking involves a combination of van der Waals and dipole-dipole interaction between the bases, which is estimated to be 4-15 kcal/mol per dinucleotide. These base stacking interactions help to minimize contact with water and are very important in stabilizing the three-dimensional structure of nucleic acids. The specificity that maintains a given base sequence in each DNA strand is contributed entirely by the hydrogen bonding between base pairs. The base-stacking interactions, which are largely non-specific with respect to the identity of the stacked bases, make the major contribution to the stability of the double helix. Under physiological conditions, double stranded DNA is more stable than are separated DNA strands. Consequently, duplex DNA predominates in vivo. Sometimes, however, the structure of duplex DNA can be disrupted, as occurs during DNA replication and transcription. Complete unwinding of a double helix and separation of the complementary single strands is called denaturation. Denaturation only occurs in vitro. Double stranded DNA can be disrupted when solutions of DNA are heated above a certain temperature or when sufficient concentrations of chaotropic agents like urea or guanidinium are added. When the temperature of the solution is raised, more and more of the bases become unstacked and hydrogen bonds between base pairs are broken. Eventually, the two strands separate completely. The temperature at which the populations of duplex of DNA and DNA with separated strands are equal is known as melting point (tm). The transition from double-stranded DNA to the single-stranded denatured form can be detected by an increase in the absorption of UV light (hyperchromic effect) or the decrease in the viscosity of the DNA solution. Each species of DNA has a characteristic melting point; higher its content of G≡C base pairs, the higher the melting point of the DNA. This is because ≡C G base pairs, with three hydrogen bonds are more stable and require more heat energy to dissociate than A=T base pairs. Structural forms of DNA Deoxyribonucleic acid is a remarkably flexible molecule. Considerable rotation is possible around a number of bonds in the sugar-phosphate backbone. In the years that followed publication of the Watson - Crick model for double-helical DNA, X-ray crystallographic studies of various synthetic oligo–deoxyribonucleotides of known sequence resulted in refinement to the structural dimensions. It is now known that DNA inside the cell does not exist only in a Watson-Crick conformation, but rather as a dynamic molecule whose exact conformation changes as the DNA strand bends in solution and is complexed to protein. DNA can assume different conformations like B-, Aand Z- forms under different physical conditions (Fig.19). Some of the key features of each DNA type are given in table 2. Table-2: Properties of A-DNA, B-DNA and Z-DNA: Property A-DNA B-DNA Z-DNA Condition in Dehydrating General Sequence of which found conditions conditions of alternating the cell purines and pyrimidines 1.Coiling Right Right handed Left handed handed 2. Rotation / + 34.7º + 34.0 º -30.0 º base pair 3. Turn 2.54 nm 3.5 nm 4.56 nm 4. Rise / base 0.23 nm 0.34 nm 0.38 nm pair 5. Base pair / 11 10.5 12 turn 6. Helical 2.3 nm 1.9 nm 1.8 nm diameter 7. Glycosyl angle C: anti G: syn C:C2'-endo, 8. Sugar pucker C3'-endo C2’-endo G: C2'-exo 9. Overall Short and Longer and Elongated morphology wide thinner and thin anti- anti- A. Features of B-DNA The Watson-Crick structure is also referred to as Bform DNA. The B-form represents the general structure of DNA in the conditions of the living cell, and is therefore, the standard point of reference in any study of the properties of DNA. This form shows right handed coiling and contains 10.5 base pairs per pitch. B. Features of A-DNA A-DNA occurs in dehydrated conditions. Hybrids between DNA and RNA, which form during transcription, are also thought to adopt the A-conformation because the hydroxyl group (2'-OH) of the ribose in RNA prevents it from adopting a B conformation made possible in DNA by the 2-deoxyribose moieties which lack the hydroxyl group. A-form DNA is also right handed helix, but the rise per base pair is 0.23 nm and the number of base pairs per helical turn is 11. A-DNA is more tightly wound than B-DNA, and the difference between the major and minor grooves are reduced. C. Features of Z-DNA Z-DNA occurs when certain sequences of base pairs are present. Certain nucleotide sequences fold up into left-handed Z-helices more readily than do others. Prominent examples are sequences in which pyrimidines alternate with purines, especially alternating C and G or 5-methyl C and G. Z-DNA is even more different from B-DNA. There are no grooves and the helix shows left-handed coiling. There are 12 base pairs per helical turn, with a rise of 0.38nm per base pair. In Z-DNA, the sugar-phosphate backbone follows a zigzagged path giving it the name Z-DNA.