In this article we will discuss about:- 1. Concept of DNA 2. Structure of DNA 3. Unusual Bases 4. Replication.
Concept of DNA:
DNA is present in the cells of all plants, animals, prokaryotes and in a number of viruses. In eukaryotes (e.g., Escherichia coli, a bacterium) the genetic material consists of a single giant molecule of DNA about 1,000 microns in length, without any associated proteins. DNA is present mainly in the chromosomes. It has also been reported in cytoplasmic organelles like—mitochondria and chloroplasts.
The DNA of all plants and animals and many viruses (polyoma virus, small pox virus, bacteriophase φ x 174, however, it is single stranded. In some viruses the genetic material is RNA. In the tobacco mosaic virus (TMV), a tobacco virus, influenza virus, however, RNA is double stranded. In bacteria and in higher plants and animals both DNA and RNA are present, viruses usually contain either DNA or RNA.
Structure of DNA:
The widely accepted molecular model of DNA is the double helix structure proposed by Watson and Crick (1953). The DNA molecule consists of two helically twisted strands connected together by ‘steps’.
Each strand consists of alternating molecules of deoxyribose (a pentose sugar) and phosphate groups. Each step is made up of a double ring purine base and a single ring pyrimidine base. The purine and pyrimidine bases are connected to deoxyribose sugar molecules.
The two strands are intertwined in a clockwise direction i.e., in a right hand helix, and run in opposite directions. The strand completes a run each 34Å. Each nucleotide occupies 3.4Å. Thus, there are 10 nucleotides per turn. Each successive nucleotide turns 36 degrees in the horizontal plane. The width of the DNA molecule is 20Å. The twisting of the strands results in the formation of deep and shallow spiral grooves.
The DNA molecule is a polymer consisting of several thousand pairs of nucleotide monomers. Each nucleotide consists of the pentose sugar-deoxyribose, a phosphate group and a nitrogenous base which may be either a purine or a pyrimidine. Deoxyribose and a nitrogenous base together form a nucleoside. A nucleoside and a phosphate together form a nucleotide.
Nucleoside = Deoxyribose + Nitrogenous base
Nucleotide = Deoxyribose + Nitrogenous base + Phosphate.
(1) Deoxyribose:
It is a pentose sugar with five carbon atoms. Four of the five carbon atoms plus a single atom of oxygen form a five-membered ring. The fifth carbon atom is outside the ring and forms a part of a-CH2 group. The four atoms of the ring are numbered 1′, 2′, 3′, and 4′. The carbon atom of —CH2 is numbered 5′. There are three —OH groups in position 1′, 3′, 5′. Hydrogen atoms are attached to carbon atoms 1′, 2′, 3′ and 4′.
Ribose, the pentose sugar of RNA, has an identical structure except that there is an —OH group instead of H on carbon atom 2′.
All the sugars in one strand are directed to one end, i.e., the strand has polarity. The sugars of the two strands are directed in opposite directions.
(2) Nitrogenous Bases:
There are two types of nitrogenous bases, pyrimidines and purines. The pyrimidines are single ring compounds with nitrogen in positions 1′ and 3′ of a six membered benzene ring. The two most common pyrimidines of DNA are cytosine (C) and thymine (T). The purines are double ring compounds.
A purine molecule consists of a 5-membered imidazole ring joined to a pyrimidine ring at positions 4′ and 5′. The two most common purines of DNA are adenine (A) and guanine (G).
Base Pairing:
Each ‘step’ of the DNA ladder is made up of purine and pyrimidine pair, i.e., of a double ring and a single ring compound. Two purines would occupy too much space, while two pyrimidines would occupy too little. Because of the purine-pyrimidine pairing the total number of purines in a double-stranded DNA molecule is equal to the total number of pyrimidines.
Thus A/T = 1 and G/C = 1 or A + G = C + T. The ratio A + T/ G + C, however, rarely equals 1, and varies with different species from 0.4 to 1.9. This ratio is commonly low in micro-organisms and high in higher animals.
The purine and pyrimidine bases pair only in certain combinations. Adenine pairs with thymine (A:T) and guanine with cytosine (G:C). The total width of the pair is 10.7Å. Adenine and thymine are joined by two hydrogen bonds through atoms attached to position 6′ and 1′.
Cytosine and guanine are joined by three hydrogen bonds through positions, 6′, 1′ and 2′. The hydrogen atom with its positive charge is shared between an oxygen atom and a nitrogen atom, both with slight negative charges. Although hydrogen bonds are weak, the fact that there are so many which gives stability to the DNA molecules. The weak hydrogen bonding enables the two strands of the DNA to separate during replication.
The pyrimidine and purine bases are linked to the deoxyribose sugar molecules. The linkage in pyrimidine nucleosides is between position 1′ of deoxyribose and 3′ of the pyrimidine. In purine nucleosides it is between position 1′ of deoxyribose and position 9′ of the purine.
(3) Phosphate:
In the DNA strand the phosphate groups alternate with deoxyribose. Each phosphate group is joined to carbon atom 3′ of one deoxyribose and to carbon atom 5′ of other. Thus each strand has a 3′ end and a 5′ end. The two strands are oriented in opposite directions. The 3′ end of one strand corresponds to the 5′ end of the other. Consequently the oxygen atoms of deoxyribose point in opposite directions in the two strands.
Unusual Bases in DNA:
Although the bases most commonly present in DNA are adenine, guanine, cytosine and thymine, other bases have also been found. In some viruses (e.g., PBS 1 and PBS 2) uracil occurs in place of thymine in DNA. Also, in some bacterial viruses (bacteriophages) cytosine is replaced by 5-hydroxymethyl cytosine (HMC). The variations of C, A and G in DNA can be considered to be the result of methylation of these bases.
Single Stranded DNA:
Although DNA of most organisms consists of two strands, single DNA is present in the bacteriophage virus φ x 174 and in several other bacterial viruses. Single strand DNA differs from double stranded DNA in the following respects.
(i) Ultra-violet absorption of double stranded DNA remains constant from 0—30°c and then rises rapidly (80oc is the critical melting point). In single stranded DNA ultra-violet absorption increases steadily from 20°c to 90°c.
(ii) Double stranded DNA is resistant to the action of formaldehyde. In single stranded DNA the reactive sites are exposed and, therefore, it is not resistant.
(iii) In double stranded DNA, A = T and G = C. In single stranded DNA of φ x 174 the proportion of A:T:G:C is 1: 1.33:0.98:0.75.
(iv) Double stranded DNA is linear while single stranded DNA is circular. During – replication, single stranded DNA becomes double stranded (replicative form).
Circular DNA:
Most of the organisms have regular double helical DNA. There are some bacteriophages and animal viruses which contain single stranded DNA. However, DNA of most phages is not linear but is in the form of a circle. The DNA of φ x 174 phages and of polyoma virus, causes cancer in monkeys and these are circular. DNA formed in the mitochondria of higher organisms is also circular.
The in vitro organ of circular DNA in the phage has been investigated. Actually DNA isolated from some viruses is linear. On heating this, the two strands of DNA separate and get denatured, Ifrecooling is done under controlled manner, this brings the two strands together and some normal double stranded DNA is obtained.
In this re-cooling some portion of DNA may take circular form also probably because the ‘sticky’ ends of DNA get exposed and the two ends on joining form a circle. By observing under electron microscope, it is found that about 20% of the re-natured DNA in polyoma virus takes circular shape during renaturation.
Replication of DNA:
One of the most important properties of DNA is that it can make exact copies of own. This process is called replication and is the fundamental basis of life. The two strands of a DNA double helix are united by hydrogen bonds between the purine and pyrimidine base pairs.
When the hydrogen bonds break, the two strands separate and unwind. The nucleus contain free nucleotides which form the necleotide pool. The nucleotides include those containing adenine, guanine, cytosine and thymine nitrogenous bases.
These free nucleotides make pairing with the nucleotides of the two separated strands by means of hydrogen bonds. Free adenine nucleotide makes pairing with the thymine nucleotide of the strand, and free guanine nucleotide with the cytosine nucleotide of the strand, etc. (A-T and G-C pairing).
In this way a new strand is formed around each old strand. The result of replication is the formation of two double helices, each similar to the original double helix.
DNA is found mostly in the chromosomes. When the chromosomes divide into two during mitosis (actually during interphase) the daughter chromosomes have same DNA double helices. Now, all the cells in the body are ultimately derived from the zygote by repeated division. It thus follows that they all have exactly similar DNA.
Replication make sure that the genes, which are segments of the DNA molecules, are present in similar sets in all cells of the body of an individual. DNA completes the requirement of a genetical material, the ability to replicate.
Outline of Replication:
1. Replication occurs during the interphase between two mitotic cycles.
2. Replication is a semi- conservative process in which each of the two double helices are formed from the parent. Double strand have one old and one new strand. Repair replication is non-conservative.
3. DNA replication requires a DNA template, a primer, deoxyribonucleoside triphosphates (dATP, dGTP, dTTP & dCTP), Mg, DNA unwinding protein, super helix relaxing protein, a modified RNA polymerase to synthesize the RNA primer, the products of dnaA, dnaB, dnaC-D, dnaE and dnaG genes and polynucleotide ligase, a joining enzyme.
4. Replication begins at a specific point called the origin.
5. According to one model replication start with a ‘nick’ or incision made by an incision enzyme (endonuclease).
6. The two strands of the DNA double helix unwind with the help of a DNA unwinding protein which binds to single DNA strands.
7. The unwinding of the strands imposes strain which is relieved by the action of a super-helix relaxing protein.
8. Initiation of DNA synthesis requires an RNA primer. The primer is synthesized by the DNA template close to the origin of replication. The synthesis is catalysed by a special form of RNA polymerase.
9. Deoxyribose nucleotides are now added to the 3′ end of the RNA primer and the main DNA strand is synthesized on the DNA template. The strand is complementary to the DNA strand and is synthesized by DNA polymerase III.
10. The enzyme DNA polymerase I now degrades the RNA primer and simultaneously catalyses the synthesis of a short DNA segment to replace the primer. This segment is then joined to the main DNA strand by a DNA ligase.
11. Replication takes place discontinuously and short pieces called Okazaki fragments are synthesized. One strand may synthesize a continuous strand and the other Okazaki fragments, or both strands may synthesize Okazaki fragments. Both new strands are synthesized in 5′ → 3′ direction. Thus, one strand is synthesized forwards and the other backwards.
12. The Okazaki pieces are joined by polynucleotide ligase, a joining enzyme, to form continuous strands.
13. Replication may be in one direction (unidirectional) from the point of origin or in the both directions (bidirectional).
Replication as A Semi-Conservative Process:
Watson and Crick were knowing that any model of DNA structure should be able to explain replication. Delbruck suggested that the Watson-Crick model of DNA could theoretically replicate by three modes, conservative, semi-conservative and dispersive.
(i) According to the conservative mode of the two double helices formed one would be of old material and the other completely of new material. Thus the old parent double helix would be unchanged.
(ii) According to the semi-conservative mode proposed by Watson and Crick, each strand of the two double helices formed would have one old and one new strand.
(iii) According to the dispersive method of replication the DNA double helix would break at several points forming many pieces. Each piece would replicate and then the pieces would reconnect at random. Thus the two double helices formed would have a patchwork of old and new pieces. 