DNA-THE DEOXYRIBONUCLEIC ACID

THE STRUCTURE AND FUNCTION OF DNA

Biologists in the 1940s had difficulty in conceiving how DNA could be the genetic material because of the apparent simplicity of its chemistry. DNA was known to be a long polymer composed of only four types of subunits, which resemble one another chemically. Early in the 1950s, DNA was examined by x-ray diffraction analysis, a technique for determining the three-dimensional atomic structure of a molecule. The early x-ray diffraction results indicated that DNA was composed of two strands of the polymer wound into a helix. The observation that DNA was double-stranded was of crucial significance and provided one of the major clues that led to the Watson - Crick Model for DNA structure. But only when this model was proposed in 1953 did DNAs potential for replication and information encoding become apparent. In this section we examine the structure of the DNA molecule and explain in general terms how it is able to store hereditary information.

HISTORY OF DNA EVOLUTION

·         In 1868: F. Miescher isolated nucleic acids from white blood cells that were acidic in nature to which he called nuclein.

·         In 1880: Fischer isolated purines and pyrimidines.

·         In 1881: Zacharis identified nuclein with chromatin.

·         In 1899: Altaman replaced the term nuclein with nucleic acid.

·         In 1900s: Kossel identified the presence of histones and protamines with nucleic acids (Nobel laurate).

·         In 1910s: P. A. Levene discovered phosphate and pentose sugars callled deoxyribose molecule.

·         In1928: Frederick Griffith demonstrate the existence of a chemical in bacteria that caries genetic information

·         In 1943: Three American Microbiologist; Ostawald Avery, Colin MacLeod and Maclyn McCarty for the first time presented the evidence that DNA is the genetic material and is made up of genes.

·         In 1944: Oswald Avery showed that degradation of DNA and not protein resulted in loss of genetic information.

·         In 1950s: Rosalind Franklin and her supervisor Maurice Wilkins were working on the X-ray diffraction model for DNA.

Rosalind Franklin (1951):

·         Generated  X-ray crystallography data suggesting a double helix with phosphates on the outside

• Rosalind Franklin who actually proposed the concept of double helix was deprived of Nobel prize due to cruel death in 1958.
• Great revolution in DNA Biology
• In 1953 February: Pauling and R. B. Corey gave a triple helix model of the DNA molecule. However he couldn’t explain the process of DNA replication.
• They were near to present about the double helix model.

• In 1953 April: J.D. Watson (an American Biologist) and F. H. C. Crick (a British Physicist) presented the double helix model of DNA (published in Nature entitled “A structure for deoxyribose nucleic acid’).
• Nobel prize awarded to

o   Watson, Crick and Wilkins

o   in 1962.

DNA STRUCTURE

DNA is composed of nitrogen bases, deoxyribose sugars and phosphate. Adenine and guanine are purine bases while cytosine and thymine are pyrimidine bases. The phosphodiester bond between sugar and phosphate molecules form the backbone of DNA. The glycosidic bond is formed between nitrogen bases and sugar molecules.

Figure 1: Nitrogen bases

Figure 2: Structure of nucleotide showing phosphodiester bond and glycosidic bond

Figure 3: Hydrogen bonding between nitrogen bases

CHARGAFF EQUIVALENT RULE

In 1948, a chemist Erwin Chargaff, on the basis paper chromatography experiment, analyze the base composition of DNA. In 1950, he discovered that: In a DNA molecule of different types of organisms, the total no. of purines is equal to the total no. of pyrimidines. A/T=G/C

Number of Purines (A+G) = Number of Pyrimidines (C+T)

WATSON AND CRICK MODEL FOR DNA

The was proposed by Watson and Crick which was published in Nature in 1953. It is also known as ‘double helix model for DNA’ molecules. However, the photograph for model was taken from X-ray diffraction photograph from Rosalind Franklin.

Figure 4: Double helix structure of DNA

According to the Model:

·         DNA molecule consists of two strands which are connected by H-bonding and they are helically twisted.

·         Each step in one strand consists of nucleotide of purine base which alternately pair with pyrimidine base.

·         DNA is a polymer of four nucleotides (A T G C).

·         Adenine pairs to thymine with 2-H bonding (A=T).

·         Gaunine pairs to cytosine with 3 H-bodings (GºC).

·         Two strands apart 20 A from each other.

·         Helix coils in right hand i.e. clockwise direction and completes at every 34 A distance.

·         Two strands are complementary to each other.

·         One strand runs 5’®3’ while the complementary strand runs 3’®5’.

·         The polarity of DNA is due to direction of phosphodiester linkage.

·         Turning results in deep and wide major groove which is the site of bonding of specific protein.

·         The distance between two strands form a minor groove.

·         One turn of double helix at every 34 A distance includes 10 nucleotides.

·         Each nucleotide is situated at a distance of 3.4 A.

·         Sugar phosphate makes the back bone of double helix of DNA molecules.

·         The DNA model also suggested a copying mechanism of the genetic material which is semi conservative in nature.

·         Experimentally proved by Mathew, Meselson and Frank W. Stahl in 1958.

·         Universally accepted.

DIFFERENT FORMS OF DNA

Three different forms of DNA are found i.e. A form, B form and Z form. The B form (10 bp/turn), which is observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions. A form (11 bp/turn), which observed under the condition of low humidity, presents in certain DNA/protein complexes. RNA double helix adopts a similar conformation.  Z form (12 bp/turn) more loosely arranged DNA is found during DNA replication.

Figure 5: Different forms of DNA

A DNA Molecule Consists of Two Complementary Chains of Nucleotides:

A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these chains is known as a DNA chain, or a DNA strand. Hydrogen bonds between the base portions of the nucleotides hold the two chains together. The nucleotides are composed of a five-carbon sugar to which is attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the name deoxyribonucleic acid), and the base maybe either adenine (A), cytosine (C), guanine (G), or thymine (T). The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a "backbone" of alternating sugar-phosphate. Because only the base differs in each of the four types of subunits, each polynucleotide chain in DNA is analogous to a necklace (the backbone) strung with four types of beads (the four bases A, C, G, and T). These same symbols (A, C, G, and T) are also commonly used to denote the four different nucleotides-that is, the bases with their attached sugar and phosphate groups. The way in which the nucleotide subunits are linked together gives a DNA strand a chemical polarity. If we think of each sugar as a block with a protruding knob (the 5'phosphate) on one side and a hole (the 3'hydroxyl) on the other, each completed chain, formed by interlocking knobs with holes, will have all of its subunits lined up in the same orientation. Moreover, the two ends of the chain will be easily distinguishable, as one has a hole (the 3'hydroxyl) and the other a knob (the 5'phosphate) at its terminus. This polarity in a DNA chain is indicated by referring to one end as 3' end and the other as the 5' end. The three-dimensional structure of DNA-the double helix-arises from the chemical and structural features of its two polynucleotide chains. Because these two chains are held together by hydrogen bonding between the bases on the different strands, all the bases are on the inside of the double helix, and the sugar-phosphate backbones are on the outside. In each case, a bulkier two-ring base (a purine) is paired with a single-ring base (a pyrimidine); A always pairs with T and G with C.

DNA TOPOLOGY

In order to fully understand DNA topology, students need to familiarize themselves with three key mathematical concepts: twist (Tw), writhe (Wr), and linking number (Lk). Twist represents the total number of double helical turns in a given segment of DNA. By convention, the right-handed twist of the Watson-Crick structure is assigned a positive value. Writhe is a property of the spatial course of the DNA and is defined as the number of times the double helix crosses itself if the molecule is projected in two dimensions. The helix-helix crossovers (i.e., nodes) are assigned a positive or negative value based on the orientation (i.e., handedness) of the DNA axis. The numerical term that describes the sum of the twist and the writhe is called the linking number, which represents the total linking within a DNA molecule. Mathematically, these properties of DNA can be expressed as:

Lk =  Tw +  Wr

Why is DNA supercoiling important? Duplex DNA is merely the storage form for the genetic information. In order to replicate or express this information, the two strands of DNA must be separated. Since the global underwinding of the genome imparts increased single-stranded character to the double helix, negative supercoiling greatly facilitates this process. As a result, replication origins and gene promoters are more easily opened, and rates of DNA replication and transcription are greatly enhanced.

While negative supercoiling promotes many DNA processes, positive supercoiling inhibits them. When tracking systems, such as replication or transcription complexes travel along the double helix, they do not spiral circumferentially around the DNA. Rather, they move linearly through the DNA and the double helix spins to accommodate this motion. Recall from the earlier discussion that the ends of chromosomal DNA are not free to rotate. As a result, the number of turns of the helix remains invariant unless the nucleic acid chain is broken. Thus, the linear movement of tracking enzymes through DNA does not change the number of turns, but merely compresses them into a shorter segment of the genetic material. Consequently, the double helix becomes increasingly overwound ahead of tracking systems. DNA overwinding, or positive supercoiling, makes it more difficult to open the two strands of the double helix and ultimately blocks essential nucleic acid processes if not alleviated.

Figure 6: DNA supercoiling

Topoisomerases I

Type I topoisomerases are denoted by “odd” numbers (topoisomerase I, III, etc.). These enzymes are monomeric in nature and require no high-energy cofactor. There are two subclasses of type I enzymes, type IA and type IB. Type I topoisomerases act by creating transient single-stranded breaks in the double helix, followed by passage of the opposite intact strand through the break (type IA) or by controlled rotation of the helix around the break (type IB). Type IA enzymes require divalent metal ions for catalytic activity and covalently attach to the 5’-terminal phosphate of the DNA. In contrast, type IB enzymes do not require divalent metal ions and covalently attach to the 3’-terminal phosphate

Topoisomerases II

Type II topoisomerases are denoted by “even” numbers (topoisomerase II, IV, etc.). These enzymes contain multiple polypeptide chains and require ATP for overall catalytic activity. Prokaryotic enzymes have an A₂B₂ structure and eukaryotic enzymes are homodimers in which the bacterial A and B subunits have merged. Based on the structure of the archetypical bacterial type II enzyme, gyrase (see below), the A subunit (or domain) contains the active site tyrosyl residue that links to DNA during the cleavage event and the B subunit (or domain) contains the site of ATP hydrolysis.

Type II topoisomerases modulate DNA topology by generating a transient double-stranded break in the DNA backbone, passing a separate double helix through the opening, and resealing the break. All bacterial and eukaryotic type II enzymes require divalent metal ions for activity and those examined so far appear to utilize a two-metal-ion mechanism similar to that of DNA polymerases and primases. The cleavage reaction of type II topoisomerases generates DNA intermediates with 4-base, 5’-cohesive ends that are covalently attached to the enzyme through their 5’-terminal phosphates