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The Machinery and the Enzymes

 In living cells, such as E. coli, the process of replication requires a set of catalysts (enzymes). The main enzyme is referred to as DNA-dependent DNA polymerase, since it uses a DNA template to catalyse the polymerisation of deoxynucleotides. These enzymes are highly efficient enzymes as they have to catalyse polymerisation of a large number of nucleotides in a very short time. E. coli that has only 4.6 ×106 bp (compare it with human whose diploid content is 6.6 × 109 bp), completes the process of replication within 18 minutes; that means the average rate of polymerisation has to be approximately 2000 bp per second. Not only do these polymerases have to be fast, but they also have to catalyse the reaction with high degree of accuracy. Any mistake during replication would result into mutations. Furthermore, energetically replication is a very expensive process. Deoxyribonucleoside triphosphates serve dual purposes. In addition to acting as substrates, they provide energy for po...
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The Experimental Proof

 It is now proven that DNA replicates semiconservatively. It was shown first in Escherichia coli and subsequently in higher organisms, such as plants and human cells. Matthew Meselson and Franklin Stahl performed the following experiment in 1958: (i) They grew E. coli in a medium containing 15NH4Cl (15N is the heavy isotope of nitrogen) as the only nitrogen source for many generations. The result was that 15N was incorporated into newly synthesised DNA (as well as other nitrogen containing compounds). This heavy DNA molecule could be distinguished from the normal DNA by centrifugation in a cesium chloride (CsCl) density gradient (Please note that 15N is not a radioactive isotope, and it can be separated from 14N only based on densities). (ii) Then they transferred the cells into a medium with normal 14NH4Cl and took samples at various definite time intervals as the cells multiplied, and extracted the DNA that remained as double-stranded helices. The various samples were separa...
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REPLICATION

 While proposing the double helical structure for DNA, Watson and Crick had immediately proposed a scheme for replication of DNA. To quote their original statement that is as follows: ‘‘It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’’ (Watson and Crick, 1953). The scheme suggested that the two strands would separate and act as a template for the synthesis of new complementary strands. After the completion of replication, each DNA molecule would have one parental and one newly synthesised strand. This scheme was termed as semiconservative DNA replication.
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Properties of Genetic Material (DNA versus RNA)

 From the foregoing discussion, it is clear that the debate between proteins versus DNA as the genetic material was unequivocally resolved from Hershey-Chase experiment. It became an established fact that it is DNA that acts as genetic material. However, it subsequently became clear that in some viruses, RNA is the genetic material (for example, Tobacco Mosaic viruses, QB bacteriophage, etc.). Answer to some of the questions such as, why DNA is the predominant genetic material, whereas RNA performs dynamic functions of messenger and adapter has to be found from the differences between chemical structures of the two nucleic acid molecules. Can you recall the two chemical differences between DNA and RNA? A molecule that can act as a genetic material must fulfill the following criteria: (i) It should be able to generate its replica (Replication).  (ii) It should be stable chemically and structurally.  (iii) It should provide the scope for slow changes (mutation) that ar...
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The Genetic Material is DNA

 The unequivocal proof that DNA is the genetic material came from the experiments of Alfred Hershey and Martha Chase (1952). They worked with viruses that infect bacteria called bacteriophages. The bacteriophage attaches to the bacteria and its genetic material then enters the bacterial cell. The bacterial cell treats the viral genetic material as if it was its own and subsequently manufactures more virus particles. Hershey and Chase worked to discover whether it was protein or DNA from the viruses that entered the bacteria. They grew some viruses on a medium that contained radioactive phosphorus and some others on medium that contained radioactive sulfur. Viruses grown in the presence of radioactive phosphorus contained radioactive DNA but not radioactive protein because DNA contains phosphorus but protein does not. Similarly, viruses grown on radioactive sulfur contained radioactive protein but not radioactive DNA because DNA does not contain sulfur. Radioactive phages were allow...
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THE SEARCH FOR GENETIC MATERIAL

 Even though the discovery of nuclein by Meischer and the proposition for principles of inheritance by Mendel were almost at the same time, but that the DNA acts as a genetic material took long to be discovered and proven. By 1926, the quest to determine the mechanism for genetic inheritance had reached the molecular level. Previous discoveries by Gregor Mendel, Walter Sutton, Thomas Hunt Morgan and numerous other scientists had narrowed the search to the chromosomes located in the nucleus of most cells. But the question of what molecule was actually the genetic material, had not been answered. Transforming Principle In 1928, Frederick Griffith, in a series of experiments with Streptococcus pneumoniae (bacterium responsible for pneumonia), witnessed a miraculous transformation in the bacteria. During the course of his experiment, a living organism (bacteria) had changed in physical form. When Streptococcus pneumoniae (pneumococcus) bacteria are grown on a culture plate, some produc...
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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 hist...
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