Deoxyribonucleic Acid: DNA
Structure of DNA:
DNA is a polymeric macromolecule consisting of many deoxyribonucleotides in a linear sequence. Four different deoxyribonucleotides are the major components of DNA which are distinguished by four nitrogenous bases.
The four bases characteristic of deoxyribonucleotides are purine derivatives:
1. adenine (A) and guanine (G)
2. pyrimidine derivatives; thymine (T) and cytosine (C).
In working out the structure of DNA, the following three types of studies have been very helpful:
1. Studies on base pairing and Equivalence:
DNA consists of two unbranched chains of polynucleotides.
The backbone of these nucleotide chains is quite regular and consists of alternate sugar and phosphate group joined with 3 and 5 phosphodiester linkages.
The sugar molecule is attached on one side with a base. In 1950, Chargaff pointed out that in DNA the amount of adenine equals the amount of thymine and the amount of guanine equals that of cytosine.
This important observation has now become a universal law that holds true for every replicative DNA found in nature. Within the limits of experimental error the ratios of A/T and G/C are equal to 1 except for the isolated ¢X174.
The ratio A+T /G+C is not equal to one and has been used to characterise the DNA from a particular source.
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2. Studies based on X-ray Diffraction:
While the work on the base composition was proceeding in Chargaff's laboratory, Rosalind Franklin and Maurice H.F. Wilkins at kings College London were using x-ray diffraction patterns for the analysis of DNA structure.
They observed that DNA from different sources had remarkably similar x-ray diffraction patterns. Astbury had earlier shown "a 3.4 Å repeat", ie., the distance between any two consecutive nucleotides in the polynucleotide chain was 3.4 A.
To this Franklin and Wilkins added a 34 A repeat which was intriguing because nothing in the polynucleotide structure appeared to correspond with this dimension. With the x-ray diagram, Franklin and Wilkins also showed that DNA molecule is a very long thin helical strand of about 20 Å diameter.
Thus, x-ray diffraction study illucidated the geometry of molecules i.e., their arrangement in space and determined the relative position of atoms making up the molecule.
The pattern obtained by Wilkins and Franklin suggested that the DNA molecule was a double stranded structure.
It was in marked contrast to the three stranded structure, Pauling had proposed earlier.
The spatial geometry of DNA was determined to be in the form of an alpha (c) helix, a spiral configuration maintaned by intramolecular hydrogen bonds.
It was then important to decide whether bases pointed outward or toward each other in the centre of the molecule.
Pauling suggested that bases pointed toward outside but Franklin felt that she had evidence that phosphate pointed toward outside and that the bases were in the centre.
3. Studies based on Titration:
The analytical studies suggested that the long nucleotide chains were held together by hydrogen bonds between base residues.
This was the situation in 1951 when James D. Watson, a 22 year old American postdoctoral research fellow, arrived in Cambridge and met Francis H.C. Crick, a physicist working for his Ph. D. degree in biophysics.
Although they were to work on different problems, they decided to collaborate on the study of DNA. In 1953, Watson and Crick proposed a double helical model of DNA molecule based on the research of Chargaff, Wilkins, Franklin, Pauling and others.
To explain their model of DNA Watson and Crick postulated several remarkable features which are as under :
1. The DNA molecule is built up of a long chain of deoxyribonucleo- tides. The chain of nucleotides is referred to as polydeoxyribonucleotide.
The sequence of nucleotides in chain makes up the primary structure of DNA.
The number of polynucleotide chains in a DNA molecule is two and not three.
2. The chains follow right handed helix with 10 bases in one complete turn of the spiral.
3. The two chains are coiled plectonemically. i.e., in an interlocked way about the same axis (Secondary structure).
4. Polynucleotide chains have polarity. The pentose sugar at one end of the chain has 5' hydroxyl or phosphoryl group (5' end) and the sugar at the other end has a 3' hydroxyl group (3' end).
One of the chains ascends and the other descends (i.e., two chains have reversed polarity or in other words of the atoms in two chains run in opposite directions).
Thus if one strand of polynucleotides phosphodiester linkages are established in 3-5' direction, the complementary strand has phosphodiester bonds in just reverse or 5'-3' direction.
In natural polynucleotides, the entire chain has polarity, i.e., all the 5' carbons point in the same direction so that the chain must end in SC at one end and 3C at the other end.
5. In the double helix the phosphates of the nucleotides are on the outside and the bases on the inside.
The nucleotides are set in the planes at right, angles to the axis of the helix and spaced at intervals of 3.4 Å or 0.34 nm. (Inm=1 nanometer = 10-9 m).
6. The two chains are held by hydrogen bonds which are established between the base pairs, or in other words bases from one polynucleotide chain are hydrogen bonded to bases from the opposite chain.
7. The pairing is highly specific because there is a fixed distance of 10 or 11 A (1.11mm) between the two sugar moieties in the opposite nucleotides Parine base normally pairs with a pyrimidine base.
Thus A-T C-G.T-A and G-C pairs are formed. A and T are joined with 2 hydrogen bonds and C and G are joined with three hydrogen bonds
There is no H-bonding between A and C or G and T. It is true that hydrogen can form only one true covalent bond but under certain conditions hydrogen which is chemically linked to one atom can form a weak linkage of non-covalent type to a second atom also.
Hydrogen bonds are much weaker than ordinary covalent bonds. It follows from the Watson and Crick model of DNA that the base sequences of the two strands are complementary.
Thus the sequence of bases in a segment of hypothetical DNA molecule might be as follows:
Watson and Crick model found immediate support from the works of Wilkins, Stokes and Wilson (1953), Franklin and Gosling (1953), Feughal- man, Langridge, Seeds, Hooper, Hemilton et al. (1955). For this brilliant piece of work Watson, Crick and Wilkins were jointly awarded Nobel Prize in 1962. Actually, this work was jointly conducted by many scientists.
Dr. Watson said sometime later,
"actually it was matter of 5 persons:Maurice Wilkins, Rosalind Franklin, Linus Pauling, F. H. C. Crick and me".
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Alternative DNA Models:
The double helix model for DNA structure proposed by Watson and Crick has been the focal point in biology for more than 20 years.
The impact and importance of the model has been so great that scientists did not feel any necessity to reexamine the x-ray diffraction data on DNA structure proposed by Watson & Crick.
It is only recently that physical scientists have begun to reexamine x-ray data interpreted by Watson and Crick and suggest some alternative conformations for DNA.
It is established beyond doubt that DNA consists of two polynucleotide chains held together by hydrogen bonds between specifically paired bases.
But the way in which the polynucleotide chains are associated has been challenged in one particularly alternative structure.
In this alternative structure DNA are viewed as side by side double helix rather than being interlocked double helix as suggested by Watson & Crick. G. A. Rodley et al. (1976) working in New Zealand and V. Chan- drasekharan et al.
In India have independently proposed a structure of B DNA which is different from the model suggested by Watson and Crick.
According to them DNA is formed of alternative right handed and left handed helices arranged side by side.
This structure has been called right-left handed helix (RL helix) or side by side double helix.
Although interlocked and side by side or RL versions of double helix are compatible with X-ray scattering and other physical data, yet side-by-side model does have one interesting feature from the genetic point of view.
There has always been some doubts about how the strand separation and unwinding of the Watson & Crick's interlocked double helix could be achieved during DNA replication.
Single stranded DNA:
DNA molecule in most organisms consists of two polynucleotide chains that are wound around each other to form a double helix. But in bacteriophage virus 6X174 and several other bacteriophages, DNA has been found to be single stranded.
Single stranded DNA differs from double stranded DNA in the following respects:
1. Double stranded DNA shows constant absorption of ultraviolet rays from 0 to 80°C (80 C° being the critical melting point) and beyond 80°C absorption rate rises rapidly. The single stranded DNA shows steady increase in absorption of UV rays from 20°C to 90°C.
2. Double stranded DNA is resistant to the action of formaldehyde whereas the single stranded DNA is not resistant because the reactive sites are exposed.
3. In double stranded DNA amount of adenine is equal to thymine and
G is equal to C. But in single stranded DNA of X174 amounts of A and G are not equal to those of Tand C respectively and the proportion is 1: 1.33 and 0.98: 0.75.
4. Single stranded DNA is circular while the double stranded is linear.
During replication single stranded DNA becomes double stranded (replicative form).
Localization of DNA in the cells:
DNA can be located in the cells in the following ways:
1. Feulgen Staining:
Feulgen (1912) found that when DNA is hy- drolysed with warm Schiff's reagent it turns reddish purple. During this reaction Schiff's reagent reacts with aldehyde groups in sugar deoxyribose. In 1924 Feulgen developed basic fuchsin staining technique which give positive indication of presence of DNA in the chromosomes.
2. Deoxyribonuclease reaction:
DNA can be removed by a specific enzyme deoxyribonuclease. After removal of DNA, the nucleus does not give the Feulgen reaction.
3. DNA absorbs ultra-violet rays at wave-length of 2600 Å. By this method it is possible to locate DNA without staining the chromosomes.
Caspersson and others have used this technique to measure nucleic acid contents of the nuclei.
Different forms of DNA:
DNA can exist in five different forms; A, B, C, D and Z forms of which B form (B DNA) is the most common of the configurations.
B DNA is a right handed double helix in which there are 10.0 base pairs per turn of the helix and base pairs are more or less perpendicular to the helix axis.
Other forms i.e., A DNA, C DNA, D DNA, and Z DNA differ from B DNA in respect of the direction of helical coiling and/or spacing and inclination of base pairs.
A DNA, C DNA and D DNA have been described both in natural and synthetic states. They are right handed double helical forms which have 11.0, 7.9-9.6, and 8.0 pairs of bases per turn respectively. In A DNA the base pairs are considerably tilted from the axis of helix at about 19° angle. Because of this displacement the depth of the deep groove is increased and that of the shallow groove decreased, axial rise being only 2.56 Å.
In C DNA, the base pairs are arranged towards the middle of the helix and are inclined at an angle of 7.8° (less than that of A DNA), D DNA, as viewed along the helix axis is not circular but hexagonal in cross section. In this form the base pairs are arranged towards the middle of the helix and are tilted at an angle of 16.7° from the axis of the helix..
Nucleic acid Hybridization:
Reannealing or renaturation of DNA is an important tool in molecular biology. It allows inter molecular hybridization between two single stranded DNAs of different species. This is called DNA/ DNA hybridization.
Since the hybridization involves parts of DNA strands which have complementary base sequances, the amount of hybridization will indicate the degree of genetic similarity.
This technique has been developed by M.Pardne and J. Gall (1970). By this technique single strand of DNA may be hybridized with RNA by complementary base pairing.
In nucleic acid hybridization experiment DNA double helices are heated So that they denature into single strands.
At this stage the denatured DNA strands are mixed with single-strand fragments of DNA or RNA obtained from other source.
Denaturation and Renaturation of DNA:
Soon after the publication of Watson and Crick's model of DNA many experiments were carried out by Warner, Rich and others using synthetic polynucleotide strands which showed that the complementary strands have marked tendency to intertwine in pairs and form right handed double helices.
Recent investigations of DNA in solution or when packed into native virus particles have shown that the double helix often departs from perfect regularity and may partially untwist to permit local separation of two polynucleotide chains (called "breathing").
The two polynucleotide strands of a DNA molecule remain held by weak hydrogen linkages.
Replication of DNA:
There are three possible methods of replication of double stranded DNA, namely:
(i) conservative
(ii) dispersive
(iii) semi-conservative
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(i) Conservative DNA replication:
In this mechanism of DNA replication two parental polynucleotide chains of DNA remain together and both the newly formed polynucleotide chains form the daughter molecule of DNA.
(ii) Dispersive DNA replication:
In this replication mechanism, the two helical strands are broken along their length and produce small fragments. Each segment of the broken strands replicates and then all become randomly connected to form two new molecules.
(iii) Semi-conservative replication:
According to Delbruck and Stent (1951) the two strands of a DNA molecule would separate from each other, maintain their integrity and both the strands will synthesize from the pool of nucleotides their complementary strands. The result would be that the newly synthesized molecules would carry or conserve one of the two strands of parent molecule.
The greatest attraction of the Watson and Crick DNA model for the geneticists is its ability to provide a simple mechanism for gene duplication.
Watson and Crick hypothesized that:
1. Before duplication, the H-bonds are broken. Then unwinding and separation of the two chains take place.
2. Then follows the formation of new complementary polynucleotide chains along the sides of old DNA strands. So two pairs of polynucleotide chains are formed from the original one pair.
In the model of replication, each newly formed double helix of DNA contains one old and one new polynucleotide strand.
According to the present day theory, the duplication of DNA in living cells including bacteria, plants and animals takes place in semi-conservative manner:
As suggested by Watson and Crick, the two strands uncoil and separate. Once the two strands are free, the base sequences which according to present view carry the genetical codes are exposed to the surrounding nuclear fluid which contains all kinds of chemical building blocks.
3. The bases of separated DNA strands now attract their complementary bases towards them.
Each in the strand would attract in correct position. the particular base that is its partner
These bases reach to the DNA strands in the form of their respective nucleoside triphosphates.
Nucleoside monophosphates and diphos phates are not directly incorporated in DNA.
Watson and Crick theory of DNA replication was tested by Messelson and Stahl in 1958. Using the isotopic and centrifugation techniques they confirmed the mechanism of DNA replication and called that type of replication as semi conservative replication because each of the two resultant daughter DNA molecules retains or conserves one parental polynucleotide strand.
The synthesis of DNA in vitro (i.e., in test tube) has been demonstrated by Kornberg and his associates in 1958. Lehman, Bessman, Sims and Kornberg (1958) purified an enzyme from the bacterium Escherichia coli. They took DNA subunits (different types of deoxyribonucleoside triphosphates), specific enzyme-DNA-polymerase and the isolated DNA strands to demonstrate the DNA synthesis in virto.
Bessman, Lehman, Sims and Kornberg (1958) have shown that in the process of polymerisation, omission of any of the four deoxyribonucleoside triphosphates retards the rate of reaction and they have also shown that no synthesis of new DNA took place in the absence of primer DNA.
The synthesis of DNA in test tube strongly supports template hypothesis of DNA replication as proposed by Watson and Crick.
This has been fully confirmed again by investigations of Professor H.G. Khorana and his associates at the Institute of Enzyme Research in the University of Wisconsin.
It is possible to make short polynucleotide chains analogous to DNA by purely chemical processes.
Thus d A (Deoxyadenylic acid) and d C (Deoxyribocytidylic acid) were induced to combine to form a dinucleotide, i.e., d AC and then six of these dinucleotides were combined to give the repeating unit d AC AC AC AC AC AC.
Replication of Bacterial DNA DNA thread of bacterial cell is regarded as bacterial chromosome or genophore or chromoneme. In the case of Escherichia coli the DNA strand contains about 3 million pairs of nucleotide bases and this is sufficient to form nearly 10,000 genes.
Replication of Viral DNA:
Viruses contain a nucleic acid and protein sheath. Nucleic acid is DNA in some and RNA in others.
Viral nucleic acid contains all the genetic informations necessary for the construction of new viruses.
In almost all the viruses genetic material appears to be a single DNA or RNA molecule, either single or double stranded, ranging in length from 1.8 µ in X 174 to 56 µ in R2, virus. DNA of T4, phage virus is circular and has a molecular weight 130/106 (ie., 200,000 nucleotide pairs) which is sufficient for 150 to 200 genes.
DNA of T, phage has about 60,000 base pairs. Coli phage lambda virus has 32 to 50 genes.
Bacterial virus ¢ X 174 contains single ring shaped DNA strand contain- ing about 5,500 nucleotides (mol. weight 1.7 X 10).
The replication process of × 174 phage virus is very remarkable and extraordinary series of changes takes place when viral nucleic acid is injected in the bacterial cell.
The normal function of the cell.e.g.. RNA and protein synthesis is disturbed.
In the host cell the viral DNA is immediately replicated and as a result of which double stranded form is obtained.
The original strand is called positive (+strand) and the newly synthesized the negative strand (-strand).
It has been found that the special protein which is produced by the virus in the early stages of infection checks the synthesis of host DNA.
The parental R.F. undergoes several rounds of semiconservative replication to produce perhaps some 15 double stranded daughter R.F. molecule. Replication of DNA can occur only at one site within the cell at any time.
The rings are packed into virus particles and lysis of the host cells occurs.
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