A possible mechanism for replication

A chemical that carries inherited information must be able to copy itself exactly. Complementary base pairing between adenine and thymine and between cytosine and guanine makes this possible.

Watson and Cricks description of DNA suggested that, during replication, the hydrogen bonds connecting base pairs are disrupted allowing the two polynucleotide chains to unwind from one another. Each chain then acts as a template for the synthesis of a new complementary polynucleotide chain. It was suggested that the DNA molecule 'unzips' from one end and new nucleotides already present in the nucleus bind with their complementary bases in each exposed chain. This therefore forms two identical molecules of DNA from the single parent molecule.

Experimental evidence

Arthur Korrnberg and his colleagues were the first to successfully replicate DNA in a test tube. They used the following ingredients:

§ intact DNA (to act as a template)

§ a mixture containing all four nucleotides

§ DNA polymerase (an enzyme which catalyses the synthesis of DNA)

§ ATP (as a source of energy).

New DNA molecules were formed, which contained the same proportions of the four bases as the original parent DNA. This was a strong indication that DNA can copy itself by complementary base pairing.

Semiconservative replication

The idea that DNA unzips before replication is an attractively simple one. This mechanism is called semiconservative replication, because each new molecule of DNA (daughter DNA) contains one intact strand from the original DNA (parental DNA) and one newly synthesised strand. However, semiconservative replication is not the only means by which DNA might replicate by complementary base pairing (figure 1).

Meselsohn and Stahl

In 1958, two American biochemists, Matthew Meselsohn and Franklin Stahl, conducted a neat experiment which gave strong support for the theory of semiconservative replication.

§ First, they grew Escherichia coli bacteria for many generations in a medium containing ¹⁵N, a heavy isotope of nitrogen. The bacteria incorporated the ¹⁵N into their DNA. This made the DNA denser than normal ('heavy' DNA).

§ A control culture of bacteria was grown in a medium with ¹⁴N, the normal, lighter isotope of nitrogen. These bacteria had normal 'light' DNA.

§ The bacteria grown in ¹⁵N were then transferred to a ¹⁴N medium and left for periods of time that corresponded to the generation time of E.. coli (about 50 minutes at 36° C).

§ Samples of bacteria were taken at intervals to analyse the parental, first-generation, and second-generation DNA.

§ The composition of the DNA was analysed using density gradient centrifugation. The mixture of the three DNA types was suspended in a solution of caesium chloride and spun at high speed in a centrifuge. The DNA separated according to its density: heavy DNA (which contained ¹⁵N) formed a band lower down the tube than the light DNA (which contained ¹⁴N). The bands became visible when the tubes were exposed to ultraviolet light.

§ The results gave overwhelming support to the semiconservative hypothesis. In the first generation, all the DNA had a density midway between that of heavy DNA and light DNA. Thus it contained equal amounts of each.

§ In the second generation, two sorts of DNA were detected: one was light DNA; the other containing equal amounts of ¹⁴N and ¹⁵N (i.e. it was like the DNA in the first-generation bacteria).

§ Throughout the investigation, DNA from the control culture produced only light bands, indicating that it contained only ¹⁴N.

§ The enzymes involved in replication

DNA replication is a complex process involving several different enzymes:

§ Helicasesseparate the two DNA strands. Their action uses energy from ATP.

§ DNA binding proteinskeep the strands separate during replication.

§ DNA polymerasescatalyse the polymerisation of nucleotides to form a polynucleotide chain in the 5' to У direction. This allows one strand to be replicated continuously.

§ The other strand is not replicated continuously but in small sections. The pieces of polynucleotide chain are joined together by an enzyme called DNA ligase.

DNA is a long molecule. DNA replication would take a long time if it started at one end and proceeded nucleotide by nucleotide along the entire length of the molecule. In fact, the double helix opens up and replicates simultaneously at a number of different sites, known as replication forks.DNA ligases then join the segments of DNA together, completing the synthesis of new DNA strands.

Quick check:

1. List the ingredients Kornberg used to make DNA in the test tube.

2. During DNA replication, what is the function of:

a) helicases

b) DNA binding proteins

c) DNA polymerase

d) DNA ligase?

3. Suppose DNA replication were conservative. What results would Meselsohn and Stahl have obtained in the first generation?

4. Describe how DNA can be made in the laboratory.

5. Interpret Meselsohn and Stahl’s experiment on semiconservative replication.

6. Describe how semiconservative replication takes place.

7. Divide the text into an introduction, principal part and conclusion.

8. Express the main idea of each part.

9. Give a title to each paragraph of the text.

10. Summarize the text in brief.

■ Text 7. The Chemical Nature Of Genes

A capsule is an outer coat covering a bacterial cell. Unlike a slime layer, it is not easily washed off. Although capsules are not essential for bacterial growth and reproduction in laboratory conditions, they can make the difference between life and death in natural situations. For example, Streptococcus pneumoniae (a member of the pneumococci, the group of pneumonia-causing bacteria used in Griffith's experiment; see text) has non-capsulated and capsulated strains. Those lacking a capsule are easily destroyed by the host and do not cause disease. However, the capsulated strain kills mice quickly. The capsule helps the bacterium resist phagocytosis by host cells. It contains a great deal of water, protecting the bacterium from desiccation; it keeps out detergents which could destroy the cell surface membrane; and it helps bacteria attach to host cells.

We know today that DNA is the chemical in which information is from parent to offspring. This spread looks at how researchers established this link between DNA and inheritance. In the 1860s, nearly 100 years before Watson and Crick's work on the structure of DNA, Gregor Mendel established that inheritance depends on factors that are transmitted from parents to offspring. In 1909 it was found that patterns of inheritance were reflected in the behaviour of chromosomes. Wilhelm Johannsen referred to these factors as genes. Genes were assumed to be located on the chromosomes because genes that are inherited together (linked genes) were found to be carried on the same chromosome. However, the chemical composition of genes was not known.

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