DNA replication

• Why does DNA replicate: DNA copies itself before cell division so that each new cell has the full amount of DNA. The method is called semi-conservative replication
• Why semi-conservative replication?

  Half of the strands in each new DNA molecule are from the original DNA molecule. This means that there is genetic continuity between generations of cells i.e. the cells produced by cell division inherit their genes from their parent cells
• A DNA molecule has a paired base structure, which makes it easy for DNA to copy itself
• The enzyme DNA helicase breaks the hydrogen bonds between bases on the 2 polynucleotide strands. This makes the helix unwind to form two single strands
• Each original strand acts as a template for a new strand. Complementary base pairing means that free-floating DNA nucleotides are attracted to their complementary exposed bases on each original template strand
• Condensation reactions join the nucleotides of the new strand together - catalysed by the enzyme DNA polymerase. Hydrogen bonds form between the bases on the original and new strands
• Each new DNA molecule contains one strand from the original DNA molecule and one new strand
• Each end of a DNA strand is slightly different in its structure. One end is called the 3' (three prime) end and one end is called the 5' (5 prime) end
• During DNA replication the active site of DNA polymerase is only complementary to the 3' end of the newly forming DNA strand - so the enzyme can only add nucleotides to the new strand at the 3' end. This means that the new strand is made in a 5' to 3' direction and that DNA polymerase moves down the template strand in a 3' to 5' direction
• Because the strands in the double helix are antiparallel, the DNA polymerase working on one of the template strands moves in the opposite direction to the DNA polymerase working on the other template strand
• Meselson and Stahl's experiment: showed DNA is replicated using the semi-conservative method. Their experiment used two isotopes of nitrogen (DNA contains nitrogen)

  1. heavy nitrogen (15N)
  2. light nitrogen (14N)
• step 1: 2 samples of bacteria were grown for many generations - one in a nutrient broth containing light nitrogen, and one in a broth with heavy nitrogen. As the bacteria reproduced, they took up nitrogen from the broth to help make nucleotides for new DNA. So the nitrogen gradually became part of the bacteria's DNA
• step 2: sample of DNA was taken from each batch of bacteria, and spun in a centrifuge. The DNA from the heavy nitrogen bacteria settled lower down the centrifuge tube than the DNA from the light nitrogen bacteria - because it is heavier
• step 3: then the bacteria grown in the heavy nitrogen broth were taken out and put in a broth containing only light nitrogen. The bacteria were left for one round of DNA replication, and then another DNA sample was taken out and spun in the centrifuge
• step 4: if replication was conservative, the original DNA, which would still be together, would settle at the bottom and the new light DNA would settle at the top
• step 5: if replication was semi-conservative, the new bacterial DNA molecules would contain one strand of the old DNA containing heavy nitrogen and one strand on new DNA containing light nitrogen. So the DNA would settle out between where the light nitrogen DNA settled out and where the heavy nitrogen DNA settled out
• step 6: as it turned out, the DNA settled out in the middle, showing that the DNA molecules contained a mixture of heavy and light nitrogen. The bacterial DNA had replicated semi-conservatively in the light nitrogen
• Once Meselson and Stahl had confirmed that DNA replication in bacteria was semi-conservative, other scientists carried out experiments to show that it was the universal method for DNA replication in all living things