Replication

We know that existing cells transfer genetic information to the new cells during cell division. What do you think will happen if each new cell receives only half the set of genetic information? Yes, the cell will not be able to function properly. This is why the DNA in an existing cells needs to be doubled (or copied) so that each new cell gets the full set of instructions. This process is DNA replication. Let’s learn more about it.

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Meselson And Stahl Experiment

Along with the double-helix structure of DNA, Watson and Crick also proposed a scheme for DNA replication. They suggested that the two DNA strands would separate and become a template for synthesis of complementary DNA strands. Therefore, each new DNA molecule would have one parental and one new DNA strand.

This scheme was referred to as semiconservative DNA replication. In 1958, Matthew Meselson and Franklin Stahl performed the following experiment to prove this mode of replication:

Experiment

• Meselson and Stahl grew E.coli on a medium that contains ¹⁵NH₄Cl as the only nitrogen source for many generations [Note: ¹⁵N is the heavy isotope of nitrogen]. As a result, all newly synthesized DNA had ¹⁵N which can be differentiated from normal DNA by centrifugation in a cesium chloride (CsCl) density gradient.
• They then transferred the cells to a medium containing normal ¹⁴NH₄Cl.
• As the cells multiplied, they collected samples at time intervals (20mins, 40mins, 60mins etc). This is because E. coli cells divide every 20 minutes. They then extracted the double-stranded DNA from the samples and separated them on CsCl gradients to measure the DNA densities.

Results

• DNA extracted after 20 minutes (one generation) in the ¹⁴NH₄Cl medium had an intermediate density. This is because it contained one parental DNA strand with the heavy ¹⁵N and one new DNA strand with the light ¹⁴N to give ¹⁵N¹⁴N.
• DNA extracted after 40 minutes (two generations) in the ¹⁴NH₄Cl medium showed equal amounts of intermediate density and light density. This is because it contained equal amounts of the hybrid ¹⁵N¹⁴N DNA (intermediate) and ¹⁴N¹⁴N DNA (light).

The Machinery Of DNA Replication

DNA Polymerase

It is the main enzyme needed for DNA replication. It uses DNA as a template to catalyze the polymerization of deoxynucleotides. DNA Polymerases should have the following properties:

• Highly efficient to carry out polymerization of a large number of nucleotides in a very short time. The process of replication in E. coli,  that has only 4.6 x 10⁶ bp (base pairs) is completed within 18 minutes. This indicates that the average rate of polymerization is approximately 2000 bp per second.
• High degree of accuracy since any mistake will lead to mutations.

Deoxyribonucleoside triphosphates

They act as substrates for polymerization. Replication is also a very energy-expensive process. Therefore, the triphosphates also provide energy for polymerization.

Origin of replication

Replication cannot start randomly at any place in DNA. It starts at a specific place in E.coli  DNA called the ‘origin of replication’.

Replication fork

Since it will require a lot of energy to separate the two DNA strands for their entire length, replication usually starts within a small opening in the DNA helix. This is the ‘replication fork’. DNA polymerase can function only in one direction 5′ → 3′. Therefore, replication of the DNA strand with polarity 3′ → 5′ is continuous.

This is also called the ‘leading strand’. While on the DNA strand with polarity 5′ → 3′, it is discontinuous and results in the formation of fragments of new DNA. This strand is the ‘lagging strand’.

DNA Ligase

This is the enzyme that joins the discontinuously synthesized fragments of DNA. In eukaryotes, DNA replication occurs in the S phase of the cell cycle. Moreover, the replication of DNA and cell division should be highly coordinated. If a cell fails to divide after DNA replication, it results in polyploidy – a condition in which a cell has more than the normal number of chromosomes.

Solved Example For You

Q1: In the Meselson and Stahl experiment, if DNA was extracted after 60 minutes in the ¹⁴NH₄Cl medium, what would be the DNA densities?

1. 75% light and 25% intermediate
2. 25% light and 75% intermediate
3. 50% light and 50% intermediate
4. 15% light and 85% intermediate

Sol: The answer is option ‘a’. After 60 minutes, i.e. three generations in the ¹⁴NH₄Cl medium, the extracted DNA will show 75% light density and 25% intermediate density.