DNA: Structure, Forms and Functions (With Diagram)

In this article we will discuss about DNA:- 1. Introduction to DNA 2. Structure of DNA Molecule 3. Nucleoside 4. Deoxyribonucleotide 5. Polynucleotide 6. Double-Helical Structure of Normal DNA 7. Different Types of DNA in Different Organisms  8. Different Forms 9. Super-Twisting 10. DNA Bending 11. Unusual Structures 12. Biological Significance or Properties 13. Functions.

Contents:

1. Introduction to DNA
2. Structure of DNA Molecule
3. Nucleoside
4. Deoxyribonucleotide
5. Polynucleotide
6. Double-Helical Structure of Normal DNA
7. Different Types of DNA in Different Organisms
8. Different Forms of DNA
9. Super-Twisting of DNA Molecules
10. DNA Bending
11. Unusual Structures of DNA
12. Biological Significance or Properties of DNA
13. Functions of DNA

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1. Introduction to DNA:

DNA or Deoxyribonucleic acid is a type of nucleic acid. It is present in all living cells of bacteria, trees, dogs, cats and human. Some viruses also contain DNA. DNA was discovered in 1868 by the German biochemist, Friedrich Miescher who called it nuclein.

At that time nobody knew that DNA is the genetic mate­rial or the molecular blueprint of life and it keeps all the precise records that pass on from generation to generation. In 1940 and early 1950s DNA’s role in determining hereditary had been confirmed by several experimental evidences.

Once DNA was recognised as the substance of heredity, research was focused on the way the DNA is chemically constructed. As a result, to study the chemical nature and the molecular architecture of DNA, several methods of extraction of highly purified DNA from a wide variety of organisms and viruses and its chemical analysis were developed.

2. Structure of DNA Molecule:

Chemical analysis of highly purified DNA have shown that it is made of four kinds of monomeric building blocks, each of which contains three types of molecules:

(i) Phosphoric acid,

(ii) Pentose sugar, and

(iii) Organic bases.

(i) Phosphoric Acid:

The phosphoric acid (H₃P0₄) is biologically called phosphate and it was discovered by Levene in 1910. Phosphoric acid (Fig. 10.1) has three reactive hydroxyl groups (—OH) of which two are involved in forming sugar phosphate backbone of both DNA and RNA. A phosphate group binds to the 5′ carbon of one and 3′ car­bon of the other neighbouring pentose sugar molecule to make the phosphate diester. The phosphate makes a nucleotide (see latter) neg­atively charged.

Therefore, a DNA becomes a polyanionic structure:

(ii) Pentose Sugar:

DNA contains β D 2′-deoxyribose sugar. It is a five-carbon sugar; hence it is a pentose sugar. Since one oxygen atom at the 2′ carbon is missing it get its name 2′-deoxy. The 2′- deoxy-containing backbone is more resistant to hydrolysis than the riboform.

D-ribose does not mean dextrorotary ribose. It is actually a form of stereoisomer [Fig. 10.2(a)]. Here the prefix D is used to refer to the configuration of sugar due to presence of asymmetric carbon atom. Any D-sugar is the mirror image of L-sugar with respect to the orientation and the position of monovalent atom or group linked with asymmetric carbon atom, i.e., monovalent atom or group in one form of sugar is just opposite in position (left- right) in other form of sugar [Fig. 10.2(b)]. In D-ribose there are three asymmetric carbon atoms.

In deoxyribose sugar, the hydroxyl group on the carbon that carries the aldehyde group can rapidly change from one position to another. The two positions are called α and β — .

Deoxyribose sugar is always present in the ring form in the structure of DNA. The ring form is derived from heterocyclic furan (C₄H₄₀) structure. The carbon atoms of the deoxyribose are numbered from the end closet to the alde­hyde and the numbers are given as 1′, 2′, 3′, 4′ and 5′ in order to differentiate them from the corresponding position in DNA bases.

It is also explained that each numbered carbon on the sugar is followed by a prime mark, therefore one speaks of 5 prime or 3 prime carbon etc.

(iii) Organic Bases:

Different types of heterocyclic nitrogen contain­ing ring compounds are found in the structure of DNA. They are called simply bases because they can combine with H⁺ in acidic solution. They are also referred as nitrogenous base due to presence of nitrogen.

I. The organic bases of DNA can be classified into two major groups:

(a) Pyrimidine’s, and

(b) Purines—on the basis of their structures.

(a) Pyrimidine’s:

Pyrimidine bases are made of a six-membered pyrimidine ring which is similar to the benzene ring except that it contains nitrogen in place of carbon at the positions of 1 and 3. Three pyrim­idine derivatives are uracil, thymine and cytosine (Fig. 10.3).

Their names are com­monly abbreviated by three capital letters such as U, T and C, respectively. While cytosine and thymine are commonly found in DNA, cytosine and uracil axe found in RNA. In RNA, thymine is replaced by uracil.

Thymine is characteristically present in DNA because thymine ensures stability of the genetic message. Otherwise retention of the uracil would result in mis-pairing and mutagenesis on subsequent replication.

Fig. 10.3 : Showing the chemical configuration on the bases, phosphates, nucleotides, base sugar linkage and sugar (Courtesy J D Watson et al)

All pyrimidine bases have a common keto- oxygen at position 2. In cytosine, an amino group (-NH₂) is present at position 4. An additional keto-oxygen is present at position 4 in uracil. But thymine contains a keto-oxygen at position 4 and a methyl group (CH₃) at position 5 (Fig. 10.3). All pyrimidine bases have a hydrogen atom at the position 1 which is involved in their linkage with carbon 1 of pentose sugar.

(b) Purines:

Purine is a derivative of pyrim­idine. It consists of a pyrimidine ring and a five-membered imidazole ring (having nitrogen at 7 and 9 position) which are fused together at 5 and 4 position.

There are two purine compounds—adenine (A) and guanine (G)—found in the structure of DNA. Adenine has an amino group (-NH₂) at 6 position while guanine has a keto group and an amino group at, 6 and 2 positions of carbon, respectively (Fig. 10.3). The pentose sugar is joined to the base by a β-N glucosidic bond between carbon atom 1 of the pentose and nitrogen atom 9 of purine bases.

(i) Rare or Minor Bases:

In addition to four common bases (ATGC), certain other unusual bases of purine and pyrimidine derivatives, called rare or minor bases, occur in small amounts in some DNA. In some viruses uracil occurs in place of thymine in DNA.

The T- even phages contain 5-hydroxy methyl cytosine (Fig. 10.4) in place of cytosine. Moreover, the hydroxyl group of hydroxyl-methyl cytosine is often glucosylated. These modifications protect the viral DNA from degradation by the host cell endonucleases.

II. General properties of bases:

All pyrimidine’s and purines have some general properties:

1. Free pyrimidine and purine bases are rela­tively insoluble in water.

2. Free pyrimidine and purine occur only in trace amounts in most cells.

3. They are weakly basic compounds that may exist in two or more tautomeric forms depending upon the pH.

4. They strongly absorb ultraviolet light in the region 250 to 260 nm. The alter­nating double and single bonds between the carbon atoms in the nitrogenous bases can interchange continuously, producing resonance. As a result, the bases absorb ultraviolet light at 260 nm. This property is very useful in the detection and quanti­tative analysis of DNA.

5. Free pyrimidine and purine bases are eas­ily separated by chromatographic or electrophoretic methods.

3. Nucleoside:

A base linked with a pentose sugar molecule is called a nucleoside, i.e., sugar + base. When deoxyribose sugar binds with base, it makes deoxyribonucleoside (Fig. 10.3). Obviously, four different kind of deoxyribo-nucleosides are found in DNA.

These are:

(a) Deoxyadenosine

(b) Deoxyguanosine

(c) Deoxythymidine, and

(d) Deoxycytidine.

As earlier pointed out, in a nucleoside C₁ , of the sugar makes β-N glycoside bond with the N₁ and N₉ of the pyrimidine and purine bases, respectively. This reaction is catalysed by synthetase enzymes and releases one —OH group from the β position of C₁ of sugar and one H atom linked the N of pyrimidine or purine.

This -OH and H combine to form one molecule of H₂O:

General properties of nucleoside:

Nucleosides show the following properties:

1. Like the free bases, free nucleosides occur only in trace amounts in most cells.

2. Nucleosides are much more soluble in wa­ter than the corresponding free bases.

3. Nucleosides are relatively soluble in alkali.

4. The purine nucleosides are rather readily hydrolysed by acid to yield free base and the pentose sugar.

5. The pyrimidine nucleosides are resistant to acid hydrolysis.

6. Both type of nucleosides are hydrolysed by specific enzyme-nucleosides.

7. They are readily separated and identified by chromatographic method.

4. Deoxyribonucleotide:

A nucleotide is derived from a nucleoside by the addition of one or more molecule of phosphoric acid. When a nucleotide is derived from de­oxyribonucleoside, it is called deoxyribonu­cleotide. The deoxyribonucleotide found in DNA, has only one phosphate group which is attached to the 5′ carbon of the deoxyribose sugar.

Therefore, the nucleotides are named deoxyadenylic acid or adenosine 5′ monophosphate (dAMP), deoxyguanylic acid or guanosine 5′ monophosphate (dGMP), deoxycytidylic acid or deoxycytidine 5′ monophosphate (dCMP) and thymidylic acid or thymidine 5′ monophosphate (dTMP) (Table 10.1).

The phosphate molecule is linked with sugar molecule to form a deoxyribonucleotide. This reaction is catalysed by phosphokinase enzyme. In this reaction, out of three hydroxyl groups of phosphate, one hydrogen atom from one hydroxyl group and one hydroxyl group from 5′ carbon of sugar come out and they combine to form a water molecule, i.e., one molecule water releases during this reaction.

In all deoxyribonucleotides, the phosphate and sugar parts are the same but they differ only due to presence of the four different bases.

General Properties of Deoxyribonucleotide:

1. Due to presence of a pyrimidine or purine base, all the deoxyribonucleotide show strong ultraviolet absorption. Fig. 10.5 gives the characteristic of molar absorption coefficient of nucleotides.

2. The phosphate groups are strong acid at pH 7.0.

3. Nucleotides are easily separated by ion- exchange chromatography.

5. Polynucleotide:

Polynucleotide chains are long, almost always un-branched polymer. A number of deoxyribonucleotides Eire covalently linked one by one to form a polynucleotide chain, i.e., deoxyribonucleotide monomer units are the building blocks of polynucleotide chain (Fig. 10.6).

The successive deoxyribonucleotide units are covalently linked by phosphodiester bridges formed between the 5′ hydroxyl group of one nucleotide and the 3′ hydroxyl group of the next. This reaction also releases one molecule of water.

Thus one by one deoxyribonucleotide joins by phosphodiester bond and, consequently, produces dimer, trimmer, tetramer and so for in polymer which is known as polynucleotide. The backbone of polynucleotide chain consists of alternating phosphates and pentose’s.

The phosphoric acid uses two of its three hydroxyl groups in the 3′, 5′ diester links. The remaining third hydroxyl group in its ionic state thus makes the strongly electronegative oxygen atom and clearly a polynucleotide is polar.

Therefore, a polynucleotide chain becomes a highly polyanionic structure. As a result the polynucleotide chain show its acid properties. This free acid group also causes nucleic acid to be highly basophilic, i.e., they stain readily with basic dyes and also enables the DNA of eukaryotic cell to form ionic bonds with basic histone proteins forming a nucleoprotein complex called chromatin.

A polynucleotide chain has a definite direc­tion. If it starts from carbon 3′ it would end in carbon 5′ and if it starts from carbon 5′ it would end in carbon 3′. Sometimes it is written as 3’→ 5′ or 5′ → 3′ or 3′ prime to 5′ prime or 5′ prime to 3′ prime.

A polynucleotide sequence containing, for example, p^cp^Gp from left to right represents p⁵‘C³‘p⁵‘G³ p. The capital let­ters denote the base and the small p denotes the phosphate within phosphoriester bond. This bond is very important and especially vul­nerable to hydrolytic cleavage—chemically and enzymatically.

6. Double-Helical Structure of Normal DNA:

In 1953 J. D. Watson and F. H. C. Crick proposed a precise three-dimensional model of DNA structure based on the X-ray crystallo­graphy data of Franking and Wilkins and the base equivalences rule formulated by Chargaff. The Watson-Crick model of DNA structure postulated that two right-handed polydeoxyribonucleotide chains or strands are coiled in heli­cal fashion around the same axis, thus forming a double helix.

The orientation of the two strands are antiparallel (Fig. 10.7), i.e., if one strand is oriented in the p-3′ → 5′-p direction, the other strand is oriented in the p-5′ → 3′-p direction. The coiling of the two strands is such that they cannot be separated except by unwinding the coils; such coiling is termed plectonemic.

In honour of this outstanding work Watson, Crick and Wilkins were awarded the Noble Prize in 1962 and one strand of double-stranded DNA is sometimes called Watson (W) and its compliment is called Crick (C). The two strands are held together by mobile hydrogen bond between the pair of bases facing each other and stacking interaction between flat aromatic surfaces of the bases.

The sugar-phosphate backbone of the two poly-de-oxribo-nucleotide chains are connected as in a ladder, on the outside, the rungs being purine and pyrimidine bases stacked on the inside of the double helix. The two antiparallel polynucleotide chains are not identical in either base sequence or composition. Instead, the two chains are complementary to each other (Fig. 10.8).

The bases on the antiparallel strands are held together in precise register: A is paired with T by two hydrogen bonds; G is paired with C by three hydrogen bonds. Neither A-G nor C-T pairs are found in DNA. Therefore, the base pair complementarity is a consequence of the size, shape and chemical composition of the base.

Beside this they are strongly hydrogen bonded base pairs than A- G and C-T pairs thus giving maximum fit and stability.

The double helix is stabilized not only by hydrogen bonding of complementary base pairs but also by stacking interactions between the flat aromatic surfaces of the bases. The latter interaction also stabilizes the heli­cal structure against the electrostatic repulsive forces between the negatively charged phos­phate groups.

This stabilising energy may be equal to or greater than that of the hydrogen bonds connecting the bases between chains. Some authors suggest that hydrophobic and Van der Waals’ interactions between adja­cent base pairs also contribute significantly to the overall stability of the double helix.

Two distinctions among the bases of de-oxy-ribonucleotides are crucial to the secondary structure of DNA. One rest on the presence of keto (C = 0) and amino (-NH₂) groups that provide opportunities for hydrogen bonding. In case of AT pairing, amino group of C6 of adenine is linked by hydrogen bond with keto group of C4 of thymine (Fig. 10.11). The inter atomic distance is 2.86 A.

In case of GC pairing, keto group of C6 and amino group of C2 of guanine are linked by hydrogen bonds with amino group of C₆ and keto group of C2 of cytosine, respectively. For these two hydrogen bonds, inter atomic distances are 2.83 A and 2.84A, respectively.

The second hydrogen bond can be formed between first nitrogen of A and third nitrogen of T in case of AT whereas, in case of GC, third bond is formed between nitrogen of the first position of both bases. The inter-atomic distances are 2.90 A in case of second bond of AT and 2.86 A in case of third bond of GC.

The bases are also set at particular angle with sugar. Both A and T are set at 51° and 50° angles with sugar molecule, respectively. But G and C make 54° and 52° angle with sugar, respectively.

The second important distinction among the bases is that they come in two sizes: the pyrimidine’s T and C are smaller than the purines A and G. However, the base pairs (ATorGC) are nearly identical in size. The AT and GC base pairs have not only the same size but very similar dimension.

Thus the two types of base pairs occupy the same amount of space (11.1 A in case of AT and 10.8 A in case of GC) allowing a fairly uniform dimension throughout the DNA double helix. These are also confirmed by X-ray crystallographic studies.

This study also reveals that the stacked bases are regularly spaced 3.4 A apart along the helix axis. The helix makes a complete turn or a pitch every 34 A, thus there are about 10 base pairs per turn and each base pair is rotated 36° relative to its neighbour.

An important element of symmetry in the helix is the rotation about the N-glycosidic bond. The conforma­tions designated anti occur more frequently in normal DNA than those conformations that are 180° opposite designated syn (Fig. 10.12). The average diameter of DNA helix is 20 A. On the outside of the molecule, the space between inter-wind strands forms a relatively narrow minor and a wide major grooves.

The right-handed double helical structure of DNA proposed by Watson and Crick is the biologically important one. It is also considered as the standard normal DNA structure which is designated as B-form DNA to differentiate it from other forms of DNA discovered later on.

7. Different Types of DNA in Different Organisms:

Studies on DNA of viruses, bacteria and eukaryotes have given rise to a number of surpris­ing findings. One is that viral and bacterial DNAs are very simple. Molecular weight is very low and approximate number of gene is relatively few. DNA is not associated with any proteins, i.e., a naked DNA thread. It is sometimes called genophore.

Second is that eukaryotic DNA is very com­plex. Molecular weight is comparatively high and the DNA contains several genes. It always makes a complex with basic histone protein.

Besides this, a number of variations still exist with respect to the structure of DNA that serves as genome material in viruses, bacteria and eukaryotic organisms.

(i) Viral DNA:

Viruses contain either DNA or genetic RNA, never both. Some of the viral DNA are double- stranded (DS) as in prokaryotic and eukary­otic cells and others are single-stranded (SS). Among them some are linear and others circular (Fig. 10.13). Hence a number of possibilities exist with respect to structure of DNA. Some of the more common viral DNA with brief description are listed in Table 10.3.

The DNAs of lamda (λ) bacteriophages and φ x 174 have received much attention. The former is a linear duplex DNA with molecu­lar weight of 32 million. The DNA’ of φ x 174 virus—which is one of the smallest DNA viruses known is circular and single-stranded with molecular weight of 1.7 million.

In 1959 Robert Sinsheimer made two important ob­servations on the DNA of 0 x 174. First the ratio of A/T and G/C were not one as is followed by the rules of base pairing in the double helix. Second, the amino groups of the purine and pyrimidine rings reacted readily with formaldehyde—a property that is generally seen only with RNA or heat dena­tured DNA.

On the basis of such observation Sinsheimer concluded that φ x 174 contains a single DNA strand. Electron microscopic studies indicate the circularity of 0 x 174 DNA. The next step in confirming the circularity of φ x 174 is that exonucleases are unable to use the intact DNA as a substrate because of a lack of free ends. This DNA contains 5,375 deoxyribonucleotide making up nine genes in the circle. Some genes are overlapping.

The linear viral DNAs show some character­istic features. The linear duplex DNA of A phage have cohesive or sticky ends. The 5′ ends of these duplex DNAs project as single-strands beyond 3′ ends. Twelve deoxyribonucleotides on these ends are complementary arid thus pair to form circular structure which can then be covalently linked by DNA ligase.

Sedimentation studies on polyoma DNA reveals that it may occur in three forms (Fig. 10.14). A supercoiled conformation, resulting from under-winding during synthesis, can be relaxed to a simple circular form if one strand of duplex DNA is severed. If both strands are severed sufficiently close together, a linear molecule results.

The liner single-stranded DNA (e.g., Provirus ) have some hairpin-like regions at each end. Besides this, SS DNA is completely devoid of random coil, base stacking, intra-strand hydrogen bonding and other types of pairing. Different forms of viral DNA molecules found in a variety of viruses are shown briefly in Fig. 10.13.

(ii) Bacterial DNA:

The bacterial cell contains a DNA that is huge compared to that present in viruses. The DNA of E. coli appears to consist of a single, enor­mous double-stranded DNA molecule with a molecular weight of about 2.8 x 10⁹, a thickness of about 2.0 nm and a contour length of about 1,360µm. E. coli DNA contains 4 million deoxyribonucleotide pairs.

This DNA is a closed and unfolded circle. Due to interaction with nucleoid proteins and RNA, the circular DNA folds into a number of loops. The loops appear to have a limited size and to number around 100 per genome. Each loop is independently supercoiled (Fig. 10.15).

Evidence in support of this comes from studies where nucleoid pro­teins are dissociated or denatured or nucleoids are subjected to RNAase treatment. In both cases, the DNA loses its supercoiled state and when RNA hydrolysis is complete, the DNA is completely unfolded. Thus nucleoid proteins and RNA appear to stabilize the supercoiled state.

In addition to the main DNA present in the nucleoid, the cytoplasm of most bacterial cell contains 1 to 20 mini circular, extra chromosomal, self-replicating, duplex DNA. These are called plasmids. The size of the plasmid ranges from 5 to 100 mega Dalton.

A number of different plasmids are able to integrate with the host chromosome; a plasmid having this ability is called an episome. Plasmids may also be transferred from one bacterial cell to another during conjugation.

Plasmids contain sufficient genetic information for their own replication. A number of host properties are specified by plasmids such as antibiotic and heavy metal resistance, nitrogen fixation, pollutant degra­dation, bacteriocin and toxin production etc.

The organisation of DNA in prokaryotes differs from that in eukaryotes in several major respects. At the gross level, eukaryotic DNA is generally linear whereas prokaryotic DNA is circular. Eukaryotic DNAs are distributed in a number of chromosomes rather than one.

Furthermore, in prokaryotic DNA, protein cod­ing genes are not split, whereas eukaryotic protein coding genes are split and the coding ‘exons’ are interspersed with generally non cod­ing ‘introns’. Unlike prokaryotes, eukaryotes have three types of RNA polymerase—each of which is responsible for the transcription of different classes of genes.

Unlike eukaryotes, almost all regulation of prokaryotes is at the level of transcription, in most cases translation of the mRNA commences before transcription is completed. Furthermore, and unlike eukaryotes genes encoding enzymes forming part of a com­mon biochemical pathway are often clustered together and coordinately regulated in operons.

The clustering of genes in this ordered man­ner, therefore, requires only a single regulatory switch for coordinate expression.

(iii) Eukaryotic Nuclear DNA:

The nuclear DNA of the eukaryotic cell is present in the nucleoplasm of a nucleus which is surrounded by a membrane. It is neither singular nor circular like bacteria. This DNA is always linear and double-stranded. Single DNA duplex makes a complex with the basic protein called histone.

This nucleoprotein complex is referred to as chromatin which is the unit of genome. The condensed form of chromatin is called chromosome. The nucleus of eukaryotic cells contains few or many chromosomes, de­pending on the species. The size of chromosome varies from species to species.

Therefore, the size of DNA molecule is also variable. The largest chromosome of Drosophila, however, contains a DNA molecule of about 4.0 cm long with molecular weight of 80 x 10⁹, nearly 40 times larger than the DNA of E. coli.

The DNA of the eukaryotic cells undergoes folding and supercoiling many times to accommodate themselves within the chromosomes. The phys­ical forms of chromosomes also varies according to the cell cycle. During interphase, they are extended or uncoiled.

During prophase, chromosomes coil and shorten until they finally reach the metaphase. During telophase they begin to uncoil and again attain the relaxed condition when the interphase of the next all cycle is reached.

Therefore, change of physical form of chromosome during cell cycle possibly brings some additional changes of folding and supercoiling pattern of DNA which is present inside the chromosome. It has been calculated that human chromosome No. 13 contains a DNA molecule about 32,000 µm long. This DNA undergoes looping and supercoiling to form a chromatid about 6 µm long and 0.8 µm in diameter.

The chromatin fibres of somatic cells are about 20 nm in diameter. In addition to DNA histone complex, chromatin also contains some acidic non-histone proteins, some enzyme pro­teins like DNA polymerase, as well as nuclear RNA and some lipids.

Rat liver chromatin has been used as a model for chromatin. It possesses a histone to DNA ratio near 1: 1, a non-histone protein to DNA ratio of 0.6 : 1 and RNA/DNA ratio of 0.1 : 1.

(iv) Eukaryotic Non-Chromosomal DNA:

Previously it was thought that DNA of the eukaryotic cell only resides in the chromosomes. But with the refinement of methodology and improvement of microscopy, it has been demon­strated quite convincingly that certain eukary­otic organ cells like mitochondria, plastids, centrioles and the yolk spherules of the egg contain DNA.

Generally, such DNAs are called cytoplasmic or non-chromosomal or extra chromosomal DNA to distinguish them from nuclear DNA. These DNAs are unique to the organelle. The structure and properties of such DNA are quite different from that of nuclear DNA. These extra-nuclear DNA molecules are capable of partial self-replication and resemble those of virus and bacteria in their circular nature.

(a) Mitochondrial DNA:

Mitochondria of eukaryotic cells contain DNA. It is always double-stranded, circular and not associated with histone or any other proteins. This DNA has some other names such as mt DNA or chondriome. There are about four to six identical copies of DNA per mitochondrion. The DNA exists within the mi­tochondrial matrix and apparently attaches at the point of inner membrane of mitochondrion.

The result of DNA-DNA renaturation and restriction endonuclease studies strongly sup­ports that all the mt DNAs in a single organism are identical. Molecular weight ranges between 10 x 10⁶ Dalton in animal and 70 x 10⁶ Daltons in higher plants.

The green algae Chlamydomonas has a linear mitochondrial genome of only 16,000 nucleotides pairs, the same size as in animals. The mitochondrial DNA of Saccharomyces cerevisiae (yeast) have been sequenced and only about one-third of them code for protein.

This finding raises the possibility that the remaining part of mtDN A of yeast contains junk DNA. In human cells, both strands of the mitochondrial DNA are transcribed at the same time from a single promoter region on each strand.

The transcripts made on one strand is called the heavy strand or H-strand because it yields huge amount of RNAs including two rRNAs, most of the tRNAs and one small poly- A containing RNA. In contrast, the light strand (L-strand) produces only eight tRNAs and one small ploy A containing RNA.

Unlike human mitochondrial genomes, plant mitochondrial genomes contain introns. In yeasts the same mitochondrial gene may have an intron in one strain but in some other strains such introns are absent. The optional introns that is present in few yeast strains are able to move in and out of genomes like transposable elements.

A small fraction of the mitochondrial DNA occurs in the form of interlocked circle which is known as catenated forms or dimers. It is suggested that catenated forms are formed during replication of mitochondrial DNA.

The role of mt DNA in the mitochondrion is similar to the role of nuclear DNA. But mt DNA cannot be expressed without assistance from the nucleus. In fact, it cannot even be replicated without such assistance. This is the level at which the molecular dependency of the mitochondrion on the nuclear genetic material is observed.

(b) Chloroplast DNA:

Chloroplasts, like mitochondria, have their own DNA (ct DNA). This DNA is generally present in multiple copies with as many as 20 to 60 ct DNAs per chloroplast. The molecular weight of ct DNA ranges from 85 x 10⁶ daltons. The length of ct DNA ranges from 40 to 60 µm. Isolated ct DNA typically exists in a variety of forms. Some are uni-circular and others appear as interlocked dimers.

Two types of dimers are found:

(i) Circular Dimers:

Which are formed by recombination between two monomers. It constitutes up to 10% of ct DNA, and

(ii) Catenated Dimers:

In which monomers interlock like links in a chain. It constitutes up to 2.5%. The monomers often appear as open chain duplex in vitro, but in situ supercoiled form is preponderate. Chloroplast DNA is made of both unique and repetitive sequences.

Repetitive sequences con­tain long repetitive region and short inverted regions (Fig. 10.16). Chloroplast DNA is large enough to code for more than 150 proteins and ribosomal RNAs (23S, 16S, 5S and 4.5S) and transfer RNA.

But, in fact, chloroplasts do not by themselves code for the synthesis of all these proteins. It is now widely accepted that both replication and differentiation are controlled partly by nuclear genome and partly by ct DNA.

(c) Yolk Spherules DNA:

In sea-urchin, DNA has been isolated from the yolk spherules. The analysis shows that the yolk contains the same amount of DNA as the mitochondria. The DNA particles in yolk are generally considered as inert storage structures.

However, there is evidence that yolk spherules are derived from mitochondria during oogenesis. Therefore it is expected that, with the transformation of mitochondria into yolk spherules, it may acquire the DNA from mitochondria. But still further experimental investigation is needed on yolk spherules DNA.

(d) Centriolar DNA:

The centrioles of the animal cells also contains DNA which is called centriolar DNA. This evidence comes from the studies by Randal and Disbrey on the ciliated Paramecium by means of fluorescence microscopy of acridine orange and autoradiography of tritiated thymidine in­corporation followed by the treatment with DNAase.

The amount of DNA per centriole is about 2 x 10⁶ gms. The DNA in centriole is not detectable shortly after cell division—possibly because it is too dispersed at the time.

8. Different Forms of DNA:

The most stable and natural form of right- handed DNA, whose structure was proposed by Watson and Crick, is also called B-form DNA. This DNA is present in almost all the living organisms. But the B-form DNA can take on other forms too under certain conditions. In all alternative forms of DNA, major changes involve helix geometry (see Table. 1(1-4).

To recall the helix geometry of B-form DNA, it is described once again:

1. The diameter of the helix is 20 A⁰.

2. The pitch, i.e., the length of helix needed to complete one turn, is 34 A⁰.

3. The distance between two base pairs is 3.4 A⁰.

4. In the physiologic solution, there are about 10.5 base pairs in one pitch rather than 10.0 found in fibre.

6. The axial rise of helix per base pair is 3.37 A⁰.

7. The tilt of a base pair is 6.3°.

Conversion of B-form DNA to other different forms of DNA takes place due to lowering of water activity, for example by addition of ethanol, by a low humidity, or by a high salt concentration. Otherwise, it retains its normal B-form without changing the physiologic con­ditions. The alternative forms have also been crystallized and subjected to X-ray structure determinations.

These forms are described below:

(i) A-DNA or Right-Handed DNA:

The A-structure is induced in DNA in 70% to 75% ethanol and is found in fibres of DNA in a dehydrated state. The primary difference between A and B helices lies in the sugar ring conformation (pucker). The sugars are C’₃-endo in the A-form but C’₂ endo in the B-form (Fig. 10.17).

This altered sugar pucker shortens the distance between adjacent phosphate on one strand by about 1A. Thus A-DNA has between 11 and 12 base pairs per helix pitch rather than the 10.5 typical of B-form.

The second major difference in A-DNA is that the base pairs are displaced from the central helix axis, towards the major grooves. The base pair in B-DNA are essentially centered over the helix axis. In the A-structure, they are shifted by side nearly 5 A from the centre, resulting in a ribbon-shaped helix with a cylindri­cal open core and a very deep but narrow major groove.

The shape of the functional groups of the bases within these grooves undoubtedly render A and B forms clearly distinguishable by protein interacting with the DNA. Base stacking—both intra and inter strand—is the key element in stabilising the A helix.

Now the helix geometry of A-DNA at a glance is:

1. The length of one pitch is 28.15 A⁰.

2. There are 11-12 base pairs per pitch.

3. The axial rise per base pair is 2.56 A⁰.

4. There is a tilting of base pairs from the axis of the helix of 20.2.

(ii) Z – DNA or Left-Handed DNA:

Z-DNA is a new family of DNA structure which is left-handed, i.e., the strands of DNA are twisted towards left-side instead of the usual right-handed twisting (Fig. 10.18). B-form is twisted clockwise whereas Z-form is twisted anti-clockwise. Therefore two possible helical forms of DNA are mirror images of each other.

Alexander Rich and his colleagues of USA have discovered the Z-form of DNA. Soon the left-handed DNA was observed in other lab­oratories, but only under certain conditions. Factors that promote the Z-structure include methylation, bromination, specific DNA bind­ing protein and sufficient torsional stress as in negatively supercoiled DNA. The Z-DNA was so-named because of the zigzag course of the backbones.

Z-DNA differs from the A and B forms not only in its left-handedness, but also in the orientation of the glycosyl bonds. The syn and anti orientations alternate, in contrast with the all-anti conformation in A- and B- DNA.

The pyrimidine nucleotides are in the standard anti conformation with a C’₂-endo sugar pucker, while the purine residues are syn and contain a C’₃-endo conformation. The Z-form is more likely to occur in alternating purine-pyrimidine sequences because steric repulsion makes it less-favourable for a pyrimidine to adopt the syn conformation.

The crystal structures have a strong dinucleotide repeat unit due to the large alternation in helix twist between the C and G in a CpG step which is about 15° while that between the G and C in the subsequent GpC is close to 45°.

Both B-DNA and A-DNA have two types of grooves—one major and one minor. But Z- DNA has the single groove which is quite deep, extending to the axis of the helix. The diameter of Z-DNA is about 18 A⁰ and the length of pith is 45 A^o.

The basic helix geometry of Z-DNA is:

1. The length of one pitch is 45 A^o.

2. Number of base pairs/pitch is 12.

3. The axial rise per base is 3.7 A^o.

4. Tilting of base pairs from the axis of helix is 7°.

5. Diameter of the helix is 18 A^o.

Z-DNA is a matter of intense current interest. It also occurs in nature. Its presence has also been established in the chromosome of the fruit-fly Drosophila melanogaster and also in the chromosomes of some other organisms.

There is growing evidence that certain DNA regions rich in guanine and cytosine within long molecules do adopt a Z-like conformation. This peculiar form of DNA is presumed to have a role in the regulation of gene activity.

It could be a recognition signal for some important function of DNA. To understand the function of Z-DNA in Drosophila melanogaster chromosomes, re­search on synthetic DNA was conducted.

It has been demonstrated that purine-pyrimidine se­quences can undergo transitions between right- handed B-conformation depending on the salt concentration or on chemical modification of the bases. The reversibility suggests that Z- DNA has some regulatory role.

For example, some nucleotide sequences can be established in the Z-form by being methylated. Methylation and de-methylation are very important in controlling the activity of genes.

There could be many possibilities by which the Z- DNA controls the gene regulations such as when certain control sites are established in the Z-form by methylation—regulatory protein binds to the site and keeps gene turned off; de-methylation might switch the site to the B- conformation, causing the regulatory molecule to let go.

Identification of Z-DNA:

Rich et al could raised antibodies by injecting rabbits with short, brominated double helices of alternating G and C nucleotides of Z-DNA. The rabbit made antibodies that were highly specific for Z-DNA and did not react at all with the B-form.

Purified rabbit antibodies for Z-DNA were used to probe biological materials for the pres­ence of Z-DNA. The polytene chromosome of D. melanogaster were treated with rabbit’s anti z-antibody and incubated to enable the antibody to bind to any Z-DNA in the chromosomes.

The unbound antibodies were washed away. Further, the bound antibodies were made vis­ible by adding a goat antibody specific for rabbit one. The goat antibody was conjugated to a fluorescent dye, so that it glowed under ultraviolet illumination. The anti z-antibody could be seen to bind to the chromosomes in a distinct and reproducible segmented pattern (Fig. 10.19).

By comparing ultraviolet micrographs that reveal the bands and inter-bands of polytene chromosomes, it was established that the flu­orescent segments are the inter-bands and not the bands. Therefore, there must be Z-DNA in the inter-bands.

It is conceivable that some Z-DNA is also present in the bands but is not visibly stained by the antibodies. Presently it appears that Z-DNA is either more abundant or more accessible in the inter bands than the bands.

(iii) C-Form DNA:

The size of helix of C-form DNA is greater than A type of DNA but is smaller than B-DNA. It is about 31 A^o. There are 9.33 base pairs per turn of the helix. There is an axial rise of base pairs of 3.03 A^o with a tilting of about 7.8° C-form is found at 66% relative humidity: in presence of Li⁺ (Lithium) ions.

(iv) D-Form and E-Form DNA:

There are some other forms of DNA called D-form and E-form found rarely as extreme variants. In case of D-form there are 8 base pairs per turn of helix. An axial rise of base pairs is 3.03 A⁰ with tilting of about 16.7°. In case of E-form, there are 7.5 base pairs per turn of helix.

Resemblances and Differences Between B-DNA and Z-DNA:

1. Both B-DNA and Z-DNA are of double helical structure.

2. In both DNAs two strands are antiparallel.

3. G Ξ C pairing is present in both B-DNA and Z-DNA.

4. B-DNA is right-handed whereas Z-DNA is left-handed.

5. The sugar-phosphate backbone in B-DNA is regular while in Z-DNA it follows zigzag course.

6. One complete helix is 34 A in B-DNA while it is 45 A⁰ in Z-DNA.

7. In Z-DNA, adjacent sugar residues exhibit → opposite orientations while in B-DNA they are in same orientation. This re­sults in dinucleotide units in Z-DNA as against mononucleotide units in B-DNA (Fig. 10.20).

8. In B-DNA there are ten base pairs at each turn while there are twelve base pairs at each turn of Z-DNA.

9. The diameter of B-DNA is 20 A⁰ whereas it is 18 A⁰ in Z-DNA.

10. Bases are more closer to the axis in Z-DNA than B-DNA.

11. The glycosidic torsion angle is anti in B- DNA and syn in B-DNA.

12. Sugar pucker is C₂ endo for B-DNA and C’₃ endo for Z-DNA in deoxyguanosine.

13. The angle of twist per repeating unit, i.e., dinucleotide is 60° as against 36° in B- DNA.

14. Base pair tilt is 6° in B-DNA whereas it is 7° in Z-DNA.

The differences mentioned above are sum­marised in Table 10.5.

9. Super-Twisting of DNA Molecules:

Normally, in a double helix, DNA consists of two polynucleotide strands that twist or wind around each other. The additional term super-twisting is generally used to describe a special feature of DNA and it means that the helical coiling is over and above that of the twisting of the two strands of the DNA double helix. Super-twisting, supercoiling and superhelicity are all interchangeable terms to define the same feature.

Initially it was thought as a special type of small circular DNA such as viral DNA, bacterial plasmids and mitochon­drial DNA. Sometimes it was believed to be an artifact of isolation.

At present, super-twisting or supercoiling is known to be an integral and extraordinarily important feature of nearly all chromosomes—whether a DNA may be circular or linear. The degree of super-twisting may be a crucial factor in stages of replication, transcription and recombination.

To understand the topology of super-twisting, suppose, two antiparallel twisted ribbons repre­sent a DNA double helix. When the free ends of such twin ribbons are joined it will simply make a ring structure. When this circular structure lies flat on a planar surface without change in the ring geometry, it is called relaxed state.

However, if before joining the free ends of the ribbon one end of the ribbon is turned through one or more 360° revolutions in the direction of unwinding, while the other end is held fixed, the ring will no longer be relaxed but, instead, will be under-wound.

This means that DNA spontaneously winds to achieve the most stable conformations—one in which all bases are in a right-handed, double helical arrangement. However, a small segment of a linear DNA is unwound or untwisted and the free ends of the two strands are linked covalently to form a closed circle.

The joining of free ends makes the DNA molecule as a whole in an unwound state. When the unwound segments pair up again to assume the double helical conformation, the entire molecule must adjust its spatial shape by twisting, for example into a right-handed super-helical form (Fig. 10.21).

An alternative deformation is for the molecule to assume a left-handed spiral. A supercoiled DNA that is under wound is called a negatively supercoiled DNA; the shape assumed is determined by the relative free energies of the various controlled forms. There are two types of negatively supercoils such as plectonemic and toroidal (Fig. 10.22).

Plectonemic supercoils are the predominant form in negatively supercoiled DNA in solution, while toroidal coiling results when the DNA is wrapped around the surface of protein such as histone to form nucleosome.

Possibly due to their interaction with proteins (histone) DNA helices in cell are unwound about 5% to 8% with respect to the standard right-handed double helical conformation of DNA in solution. When DNA is removed from cells and stripped off its protein, it becomes negatively twisted.

If, instead, a linear DNA is twisted more tightly than normal before the free ends are joined together, an over-wound or positively supercoiled DNA is formed. A positively supercoiled DNA can assume the form of a left-handed superhelix or a right-handed spiral (Fig. 10.21).

Some enzymes are also involved during su­percoiling and relaxing state of DNA. Topoisomerases directly change the super helical state of DNA. Gyrase, an enzyme, induces negative supertwists and relaxing enzymes remove them.

10. DNA Bending:

Long chains of DNA are more flexible than the short pieces of DNA (about 100 bp). Short chains of DNA are comparatively stiff and it is very difficult to bend them. However, certain sequences and structures present in DNA molecule generate an intrinsic bend and are known as bendable DNA segments.

Such segments are inherently flexible. Bent segments are important features in regions of DNA that control replication, transcription and recombi­nation.

Each mini circle of kinetoplast DNA (K- DNA) of Trypanosomes contains a small region of bendable DNA sequence. Polyacrylamide gel electrophoresis (PAGE) can be used as a tech­nique for identifying DNA with a bend.

When DNA is cut into a number of small fragments by restriction digestion, then the fragments with the bend in the centre will move in the PAGE slowly while the fragments with the bend at the end move very fast. Therefore, on the basis of rate of mobility, the locus of bending can be defined by PAGE of DNA fragments that are more or less identical in length (Fig. 10.23).

A bend, when it is a part of a longer molecule, creates a tight loop and is induced by adenine repeats. Although bends are caused by adenine repeats, thymine dimers, insertion of one or a few base pairs in one strand of the duplex, also bend DNA.

There are some non-intrinsic bends which are formed by the influence of a bound protein. Bending of DNA around a protein surface was first seen in a nucleosome in which at 145 bp segment is wrapped nearly twice around a histone octamer and the segment bends more than 600°. Due to tight wrapping, the grooves of the DNA must be narrowed along the surface facing the protein and widened on the outside edges.

Many non-histone proteins also induce bend­ing of DNA.

These are:

(a) Catabolic activa­tor protein (CAP) and the galactose repressor

(b) RNA polymerase of E. coli

(c) Recombi­nation enzymes such as Tn 3 resolvase and A integrase

(d) Restriction enzymes Eco RI

(e) The small DNA binding proteins IHF, HU and Fis and (f) DNA gyrase.

The magnitude of protein induced bends is variable and ranges from 30° to 140°

Functions of DNA Bending:

The possible functions of DNA bending are:

1. Condensation and packaging of DNA.

2. Bringing together distant binding sites in linear DNA.

3. Formation of special protein-DNA struc­ture.

4. Straining the DNA helix to stimulate its cleavage or melting.

11. Unusual Structures of DNA:

In addition to bending, the diversity of DNA can be expressed in unusual DNA forms. Such forms include cruciforms, intra-molecular triplex and single-stranded DNA bubbles. Z-DNA is also included as unusual structure. Physical, chemical and enzymatic probes can be used to identify unusual DNA structures in vitro but are difficult to apply in vivo.

(i) Cruciforms:

The cruciform means a ‘cross’ or ‘plus sign’ like structure. It is made of two exposed hairpins. Cruciforms may develop at the site of a palin­dromic DNA sequence (the standard definition of a palindrome is a word or sentence that reads the same forward and backward) an inverted repeat with a twofold axis of symmetry.

Two distinct pathways have been suggested for cruciform extension. These are:

1. Most commonly, the little central part of a palindromic sequence or an inverted repeat opens and forms a central bub­ble by melting of a few base pairs of the duplex (Fig. 10.24). This is followed by intrastrand pairing which is known as proto-cruciform. The proto-cruciform is converted into fully-extended cruciform by the process of branch migration of the junction.

2. In a less common pathway, the whole segment of a palindromic sequence opens by the melting of a large region of DNA to form a single-stranded DNA bubble (Fig. 10.24). This is followed by intrastrand pairing to create the stems of the hairpins.

(ii) Intra-Molecular Triplex or H-DNA:

DNA may contain some repeated segments of pyrimidine’s and purines such as (TC)n paired with (AG)n. These segments can form a three stranded helix or H-DNA. The triplex is formed when half of the normal duplex is disrupted and the poly-pyrimidine strand folds back and inserts itself into the major groove of the remaining duplex (Fig. 10.25). The poly-pyrimidine strand involved in triplex struc­ture is known as donor.

The donated strand associates by Hoogsteen base pairing with the purines in the duplex region without disrupting the conventional Watson-Crick pairs. Formation of Hoogsteen base pairs requires protonation of the C residues and a pH of 5 stabilizes the structure.

The requirement of H+ ions for protonation gives its ncune H-DNA. The triplex is a right-heinded helix and has a loop of about four nucleotides in the poly-pyrimidine strand at its tip. The base forms a flexible hinge and, hence, its synonym is hinge DNA. The poly-purine strand of the disrupted helix remains unpaired.

Formation of the triplex is driven by the removal of negative supercoils. Long repeats remove more supercoils and require lower super-helix densities for triplex formation. Re­peats longer than 30 base pair remove about one super-helical turn for every 11 base pairs participating in the structure.

Thus, both disruption of the duplex and formation of the triplex must contribute to unwinding. If melt­ing of the duplex to generate the only source for relaxing the supercoils, then removal of only half this number of turns would be expected. The triplex region does not contribute to the linking number of the molecule because it has an equal number of strand crossings in both the positive and negative directions.

The triple-stranded DNA shows the following characteristic features:

1. The exact alternating nucleotide sequence rather than simply the one purine-rich and one pyrimidine-rich strand is key as hydrogen bonding between the strands is essential.

2. Minor disruption in the repeats or 1-2 base pair mutation will not block triplex for­mation providing enough repeat units are present to stabilize a structure containing the mismatches. Otherwise two shorter triplexes form as a result of a 3 base pair disruption in a long repeat. Nucleotide insertions in the middle of the repeats are much less disruptive because they can be accommodated within the loop.

3. A high GC content stabilizes the structure. An-Tn repeats fails to form triplexes but (AG)n (CT)n and Gn-Cn do so efficiently.

4. Sequences which form triplexes in vitro have been isolated from the regulatory region of genes, suggesting a possible role in gene expression. However, direct evidence for the presence of triple- stranded DNA in the cell is still lacking.

(iii) Single-Stranded DNA Bubbles:

Negatively supercoiled DNA has a more single- stranded character. This character is readily apparent from its under wound nature. The degree to which under winding is separated into single-stranded regions, negative super-helical twists depends on the DNA sequence, solution conditions and the presence of proteins.

How­ever, some regions of DNA have an especially high tendency for adopting a single-strand, melted configuration.

Two portions of DNA that are unusually stably unwound when present on a supercoiled circle are known as single-stranded DNA bubble (Fig. 10.26) and have been identified by the action of single-strand specific nucleuses and by the removal of negative supercoils. Single-stranded DNA bubble regions are very A-T rich.

Such DNA unwinding segments about 100- 120 nucleotides long are present on the com­monly used plasmid pBR322.

However, similar unwinding elements have been identified in the 13 bp repeated elements of the E. coli chromosomal origin sequence, Ori C and in the autonomously replicating sequence (ARC) of the yeast 2µ plasmid where they may play a key role in destabilising the duplex during initiation at the origin of chromosome replication.

(iv) Intermolecular Structures:

DNA may contain several intermolecular struc­tures such as R-loops, D-loops, Par-anemic joints, Holliday junctions, knots and catenanes.

These unusual structures are de­scribed below:

(a) R-Loops:

If double-stranded DNA is treated with a 50% solution of form-amide at room temperature or 25°C, some hydrogen bonds between the strands of the molecule break, weakening but not completely melting the duplex.

If RNA that is complementary to one strand of the duplex DNA is introduced, the RNA binds to its complementary site on one DNA strand, displacing the other DNA strand. This occurs because RNA-DNA duplex is more stable than a DNA-DNA duplex. The hybrid duplex and the displaced stretch of single-stranded DNA are called an R-loop.

The two R-loops result from the hybridisa­tion of 18S and 25S ribosomal RNA from yeast with a region of bacteriophage A DNA that contains an inserted stretch of yeast ribosomal genes. The association of a region of RNA with a negatively supercoiled duplex is stabilized by the removal of negative super-helical turns.

R-loops form temporarily during transcription and require RNA polymerase or some other enzyme to displace them. R-loop formation during transcription may be essential for DNA replication to serve as a primer for plasmid Col E_i and phage T₇ DNA or as an activator for Col E₁ and E. coli Ori C.

(b) D-Lopp:

Like R-loop, D-loops are formed when additional single-strands of DNA are taken up by the duplex. Formation of a D-loop is facilitated in negatively supercoiled DNA and can occur spontaneously. Although D-loops are formed in a high percentage of mammalian mitochondrial DNAs, they are most prominent as intermediates in genetic recombination in which their formation is catalysed by strand- exchange enzymes.

(c) Par-Anemic Joints:

Par-anemic joints are protein stabilized struc­tures in which the additional single-stranded DNA pairs with its complement in the melted portion of duplex but is not stably inter-winded as in the classical helical duplex.

This lack of net topological inter-winding enables a par-ane­mic joint to be formed without participation of a free end on either DNA molecule and to take place between a single-stranded and a duplex circle.

The length of ss DNA that is able to pair with the duplex circle appears to be limited by the superhelicity of the duplex which is relaxed by joint formation. The structure of the DNA strands within the par-anemic joint that allows pairing over several hundred base pairs in the absence of net helical inter-winding has not been satisfactorily established.

There are two possibilities:

(i) Unwinding of the duplex DNA so that one of its strands can pair side by side with the incoming strand;

(ii) Accommodating alternating segments of right and left-handed helics across the joint region involving no net change in the total number of helical turns introduced.

(d) Plectonemic Joints:

A free end on the DNA strand or the action of a topoisomerase allows intertwining of the strands of a par-anemic joint, converting it to the fully stabilized helical plectonemic joint.

(e) Holliday Junctions:

These junctions between four DNA strands are important intermediates in genetic recombina­tion. Enzymes specific for these forked DNAs are encoded by phages T₇ and T₄ as well as by yeast. Formation of Holliday junction takes place through some steps.

In the first step, a nick is made in one strand of each of the two sets of duplexes that are going to recombine. Strand exchange then occurs at the site of the nick, producing a crossed-strand Holliday Junc­tion (Fig. 10.27).

Breaking of the hydrogen bonds within the parental duplexes followed by the exchange of strands and reformation of hydrogen bond leads to branch migration. Holliday junctions, in fact, exhibit two-fold symmetry—the arms pair with each other, forming two sets of collinear helices.

This collinear conformation allows stacking between the bases across the junction of the arms. The identity of the final bases at the four-way junction of the arm determines which arms will pair with each other to provide the most stable conformation. The angles between the two sets of collinear helices are two acute and two obtuse angles.

(f) Knots and Catenanes:

In knotted and catenated forms, the DNA duplex is interlinked and intra-molecular linkage forms a knot while linkage between two circles generates a catenane. Sometimes interlocked DNA forms are often the products of replication and recombination on circular DNA molecules, although in most cases they are short-lived intermediates.

Multiple inter-wined catenated dimers are a common late intermediate in the replication of circular DNAs and may occur as well in the topology called constrained domains of linear DNA.

Helical inter-wines between the two tem­plate strands can be converted into interlocks between the already replicated duplex portions of the molecule, allowing replication to be com­pleted. For an instance, 100 base pairs of the template to be replicated could be converted into ten intermolecular inter-wines between the daughter circles, which could be subsequently removed by topoisomerase.

Catenanes are involved in the replication of many circular genomes including SV 40 animal virus and the small E. coli plasmids. The behaviour of topoisomerase mutants in yeast suggests that similar structures are seen in the replication of large linear chromosomes.

Knots and Catenanes are useful in defining the mechanisms by which certain transactions of DNA occur:

(i) Type I and type II topoisomerase can be distinguished on the basis of their ability to untie knots in completely duplex DNA, because type II enzymes alone are active on such molecules.

(ii) Interlocked DNA circles can also be used to determine whether a DNA-binding protein interacts with two distantly located on a single circle by linear tracking along the DNA or by loop formation between the sites looping can occur both intra-molec­ularly and on catenated circles, whereas tracking is impossible between the two circles in a catenane.

(iii) Determining whether or not catenate in­terlocks can replace the requirement for DNA superhelicity provides important in­sight into how the energy of supercoiling is exploited by a reaction.

(iv) The structure of a knotted DNA product of a recombination reaction gives information about the organisation of the synaptic complex.

(v) The study of DNA interlocks represents an additional approach to an understanding of the dynamics of DNA transactions.

12. Biological Significance or Properties of DNA:

DNA has several biological significance partic­ularly in the cellular environment and in the organism as a whole.

It shows the following properties:

1. It replicates itself accurately during cell growth and duplication. This is known as auto synthesis.

2. It has the potentiality to carry all kinds of necessary biological information and is involved in gene action which—through a series of chemical reactions results in the ultimate expression of characteristics within the organism. The latter property is known as hetero-catalysis.

3. Its structure is fundamentally stable so that heritable changes (mutation) occur only very rarely.

4. It transmits all necessary biological infor­mation to the next cell generation or the next generation of the organism.

5. In most of the organisms DNA is the genetic material.

6. DNA must show a very wide diversity in form to account for the innumerable diversity in the characters of organisms occurring in nature.

13. Functions of DNA:

The structure of DNA is highly rationalised as a design to fulfil several primary functions efficiently as follows:

1. Primarily DNA acts as the cell’s genetic blueprint which is mainly used for tran­scription of the encoded information and to serve as the template for replication.

2. DNA must remain stable enough to allow on a modest level of change over many generations.

3. The linear array of four nucleotides, arranged in a defined order, allows the molecule to encode an essentially endless and diverse amount of information.

4. The double helix structure of DNA main­tains two copies of the information at all times. If lost by errors in replication or by DNA damage, the information is often retrievable from an undamaged comple­mentary strand.

5. Complementarity in the nucleotide sequence provides .the basis for the transcription into the temporary but directly usable mRNA and for replication prior to cell division.

6. Base complementarity, melting and annealing of the helix and its ability to accommodate at least transiently alien or new DNA molecule provide the foundations for recombination between DNA molecules.

7. Recombination is essential for most path­ways of damage repair, for replication of the ends of some linear molecules and for the initiation of replication.

8. Recombination provides for the communi­cation between DNA molecules in a pop­ulation, across species and even across kingdom boundaries, affording a means for maintaining both genetic stability and diversity.

9. Finally, super twisting, bending and topo­logical flexibility inherent in DNA play a key role in packing these long molecules into a cell that is only a tiny fraction of their length.