James Dewey Watson was born in Chicago on 6 April 1928. In 1947, he received B.Sc. degree in Zoology. During these years his interest in bird-watching had matured into a serious desire to learn genetics. This became possible when he received a Fellowship for graduate study in Zoology at Indiana University, Bloomington, where he received his Ph.D. degree in 1950 on a study of the effect of hard X-rays on bacteriophage multiplication.
He met Crick and discovered their common interest in solving the DNA structure. Their first serious effort, was unsatisfactory. Their second effort based upon more experimental evidence and better appreciation of the nucleic acid literature, resulted, early in March 1953, in the proposal of the complementary double-helical configuration.
Francis Harry Compton Crick was born on 8 June 1916, at Northampton, England. He studied physics at University College, London and obtained a B.Sc. in 1937. He completed Ph.D. in 1954 on a thesis entitled “X-ray Diffraction: Polypeptides and Proteins”.
A critical influence in Crick’s career was his friendship with J. D. Watson, then a young man of 23, leading in 1953 to the proposal of the double-helical structure for DNA and the replication scheme. Crick was made an F.R.S. in 1959. The honours to Watson with Crick include: the John Collins Warren Prize of the Massachusetts General Hospital, in 1959;
The Lasker Award, in 1960; the Research Corporation Prize, in 1962 and above all, the Nobel Prize in 1962.
DNA is a long polymer of deoxyribonucleotides. The length of DNA is usually defined as number of nucleotides (or a pair of nucleotide referred to as base pairs) present in it. This also is the characteristic of an organism. For example, a bacteriophage known as f ×174 has 5386 nucleotides, Bacteriophage lambda has 48502 base pairs (bp), Escherichia coli has 4.6 × 106 bp, and haploid content of human DNA is 3.3 × 109 bp. Let us discuss the structure of such a long polymer.
6.1.1 Structure of Polynucleotide Chain
Let us recapitulate the chemical structure of a polynucleotide chain (DNA or RNA). A nucleotide has three components – a nitrogenous base, a pentose sugar (ribose in case of RNA, and deoxyribose for DNA), and a phosphate group. There are two types of nitrogenous bases – Purines (Adenine and Guanine), and Pyrimidines (Cytosine, Uracil and Thymine)Cytosine is common for both DNA and RNA and Thymine is present in DNA. Uracil is present in RNA at the place of Thymine. A nitrogenous base is linked to the pentose sugar through a N-glycosidic linkage to form a nucleoside, such as adenosine or deoxyadenosine, guanosine or deoxyguanosine, cytidine or deoxycytidine and uridine or deoxythymidine. When a phosphate group is linked to 5′-OH of a nucleoside through phosphoester linkage, a corresponding nucleotide (or deoxynucleotide depending upon the type of sugar present) is formed. Two nucleotides are linked through 3′-5′ phosphodiester linkage to form a dinucleotide. More nucleotides can be joined in such a manner to form a polynucleotide chain. A polymer thus formed has at one end a free phosphate moiety at 5′-end of ribose sugar, which is referred to as 5’-end of polynucleotide chain. Similarly, at the other end of the polymer the ribose has a free 3′-OH group which is referred to as 3′ -end of the polynucleotide chain. The backbone in a polynucleotide chain is formed due to sugar and phosphates. The nitrogenous bases linked to sugar moiety project from the backbone (Figure 6.1).
In RNA, every nucleotide residue has an additional –OH group present at 2′-position in the ribose. Also, in RNA the uracil is found at the place of thymine (5-methyl uracil, another chemical name for thymine). DNA as an acidic substance present in nucleus was first identified by Friedrich Meischer in 1869. He named it as ‘Nuclein’. However, due to technical limitation in isolating such a long polymer intact, the elucidation of structure of DNA remained elusive for a very long period of time. It was only in 1953 that James Watson and Francis Crick, based on the X-ray diffraction data produced by Maurice Wilkins and Rosalind Franklin, proposed a very simple but famous Double Helix model for the structure of DNA. One of the hallmarks of their proposition was base pairing between the two strands of polynucleotide chains. However, this proposition was also based on the observation of Erwin Chargaff that for a double stranded DNA, the ratios between Adenine and Thymine and Guanine and Cytosine are constant and equals one.
The base pairing confers a very unique property to the polynucleotide chains. They are said to be complementary to each other, and therefore if the sequence of bases in one strand is known then the sequence in other strand can be predicted. Also, if each strand from a DNA (let us call it as a parental DNA) acts as a template for synthesis of a new strand, the two double stranded DNA (let us call themas daughter DNA) thus, produced would be identical to the parental DNAmolecule. Because of this, the genetic implications of the structure of DNA became very clear.
The salient features of the Double-helix structure of DNA are as follows:
(i) It is made of two polynucleotide chains, where the backbone is constituted by sugar-phosphate, and the bases project inside.
(ii) The two chains have anti-parallel polarity. It means, if one chain has the polarity 5’à3′, the other has 3’à5′ .
(iii) The bases in two strands are paired through hydrogen bond (H-bonds) forming base pairs (bp). Adenine forms two hydrogen bonds with Thymine from opposite strand and vice-versa. Similarly, Guanine is bonded with Cytosine with three H-bonds. As a result, always a purine comes opposite to a pyrimidine. This generates approximately uniform distance between the two strands of the helix (Figure 6.2).
(iv) The two chains are coiled in a right-handed fashion. The pitch of the helix is 3.4 nm (a nanometre is one billionth of a metre, that is 10-9 m) and there are roughly 10 bp in each turn. Consequently, the distance between a bp in a helix is approximately equal to 0.34 nm.
(v) The plane of one base pair stacks over the other in double helix. This, in addition to H-bonds, confers stability of the helical structure (Figure 6.3).
Compare the structure of purines and pyrimidines. Can you find out why the distance between two polynucleotide chains in DNA remains almost constant?
The proposition of a double helix structure for DNA and its simplicity in explaining the genetic implication became revolutionary. Very soon, Francis Crick proposed the Central dogma in molecular biology, which states that the genetic information flows from DNAà RNAà Protein.
In some viruses the flow of information is in reverse direction, that is, from RNA to DNA. Can you suggest a simple name to the process?
6.1.2 Packaging of DNA Helix
Taken the distance between two consecutive base pairs as 0.34 nm (0.34×10–9 m), if the length of DNA double helix in a typical mammalian cell is calculated (simply by multiplying the total number of bp with distance between two consecutive bp, that is, 6.6 × 109 bp × 0.34 × 10-9m/bp), it comes out to be approximately 2.2 metres. A length that is far greater than the dimension of a typical nucleus (approximately 10–6 m). How is such a long polymer packaged in a cell?
If the length of E. coli DNA is 1.36 mm, can you calculate the number of base pairs in E.coli?
In prokaryotes, such as, E. coli, though they do not have a defined nucleus, the DNA is not scattered throughout the cell. DNA (being negatively charged) is held with some proteins (that have positive charges) in a region termed as ‘nucleoid’. The DNA in nucleoid is organised in large loops held by proteins.
In eukaryotes, this organisation is much more complex. There is a set of positively charged, basic proteins called histones. A protein acquires charge depending upon the abundance of amino acids residues with charged side chains. Histones are rich in the basic amino acid residues lysines and arginines. Both the amino acid residues carry positive charges in their side chains. Histones are organised to form a unit of eight molecules called as histone octamer. The negatively charged DNA is wrapped around the positively charged histone octamer to form a structure called nucleosome (Figure 6.4 a). A typical nucleosome contains 200 bp of DNA helix. Nucleosomes constitute the repeating unit of a structure in nucleus called chromatin, thread-like stained (coloured) bodies seen in nucleus. The nucleosomes in chromatin are seen as ‘beads-on-string’ structure when viewed under electron microscope (EM) (Figure 6.4 b).
Theoretically, how many such beads (nucleosomes) do you imagine are present in a mammalian cell?
The beads-on-string structure in chromatin is packaged to form chromatin fibers that are further coiled and condensed at metaphase stage of cell division to form chromosomes. The packaging of chromatin at higher level requires additional set of proteins that collectively are referred to as Non-histone Chromosomal (NHC) proteins. In a typical nucleus, some region of chromatin are loosely packed (and stains light) and are referred to as euchromatin. The chromatin that is more densely packed and stains dark are called as Heterochromatin. Euchromatin is said to be transcriptionally active chromatin, whereas heterochromatin is inactive.