DNA Replication

DNA Replication

Since DNA forms the genetic code and that it is known that genes may be inherited, it follows that DNA must be copied exactly before being incorporated into gametes at meiosis.

It also follows that all new cells in an organism must gain a copy of the genes at mitosis, because they are able to continue the characteristic biochemical behaviour of that organism.

What is the mechanism for this exact copying or replication of the DNA?

Three theories existed:

  1. The parent DNA molecule breaks into segments and new nucleotides fill in the gaps precisely (fragmentation theory).
  2. The complete parent DNA molecule acts as a template for the new daughter molecule, which is assembled from new nucleotides. The parent molecule is unchanged (conservative hypothesis).
  3. The parent DNA molecule separates into its two component strands, each of which acts as a template for the formation of a new complementary strand. The two daughter molecules therefore contain half the parent DNA and half new DNA (semi-conservative hypothesis).

The semi conservative hypothesis was shown to be the true mechanism by the work of Meselsohn and Stahl (1958).

In their experiment they grew the bacterium E.coli in the presence of radioactive 15N until a culture was obtained in which all the DNA was labelled with 15N.

A subculture of this labelled bacterium was than transferred for growth in the presence of normal 14N. The generation time of E.coli is known, so it was possible to take samples of this growing subculture after exactly one, two, three generations and so on.

The semi conservative hypothesis

Each sample had its DNA extracted and the isolated DNA was then centrifuged in a caesium chloride solution (high viscosity) at 40,000G for 20 hours, causing the DNA to sediment out.

The heavier the DNA, the further it moved down the centrifuge tubes. 15N DNA is heavier than 14N DNA. Mixed 14N and 15N DNA is intermediate in mass between the two.

The original 15N DNA moved to the lowest position in the tube.

After one generation all the DNA moved to an intermediate position, indicating the presence of only mixed 14N and 15N DNA. This was because the DNA in this generation contained one strand of the parent molecule and one new 14N strand.

Had the conservative hypothesis been true, two DNA masses would have been visible, one heavy and the other light.

In the second generation half the DNA was intermediate and half was light, for the same reason.

The semi conservative hypothesis

The actual process is simple. To begin with one strand in the DNA duplex is nicked by the enzyme DNA topoisomerase, allowing part of the molecule to unravel to form a replication fork (the DNA is replicated a bit at a time and the whole molecule is never completely uncoiled).

Process of DNA replication

Next, the enzyme DNA helicase splits the two strands by breaking the hydrogen bonds. This exposes the bases.

Process of DNA replication

DNA polymerase enzyme then moves along the exposed bases sequences, creating a new complementary strand as it goes. DNA polymerase reads the exposed code from the 3' to the 5' end and therefore assembles the new strand from the 5' to the 3'.

Several molecules of DNA polymerase act simultaneously, each assembling a separate section of the new strand of DNA. Each DNA polymerase is preceded by an RNA polymerase enzyme, which constructs an RNA primer to guide the action of the DNA polymerase.

These new DNA segments are then joined together by the enzyme DNA ligase. The two new daughter molecules then coil up again to reform the double helix structure. This process occurs during the S phase of the cell cycle.

Process of DNA replication