Nucleic Acids

DNA Replication

In order for new cells to be created all the DNA in a cell must be replicated first, to ensure that when the cell splits in half each new cell still contains the full amount of DNA. This occurs in S-phase in interphase of the cell cycle.

Nucleic Acids, figure 1

When describing the DNA double helix, the top and bottom of each strand is described as either the 3” (prime) end or the 5” (prime) end. This number refers to which carbon within the deoxyribose sugar of the nucleotide is closest to the top/bottom – see diagram below. This is relevant, as an enzyme that catalyses DNA replication is complementary in shape to the 3” end, and can therefore only attach to the DNA at this location.

Nucleic Acids, figure 2

Stages of DNA Replication

Before the DNA can be copied the double helix must first unwind. The enzyme DNA helicase breaks the hydrogen bonds between the complementary bases of the two DNA polymers within the double helix. This causes the double helix to unwind and the two strands to separate, or unzip. These two separated strands both act as templates for DNA replication.

The point at which the unzipping stops is called the replication fork. Not all the DNA is unzipped in one go as this increases the chances of copying errors resulting in mutations.

Within the nucleus there are free floating DNA nucleotides. If a free floating DNA nucleotide aligns next to a complementary base on either template strand of DNA then hydrogen bonds will form between them.

The enzyme DNA polymerase is responsible for then forming the phosphodiester bond between these nucleotides to create a new polymer chain of DNA. DNA polymerase can only attach at the 3” end, and therefore will move along the template strand in the 3” to 5” direction.

When the enzyme is moving towards the replication fork, the new strand is referred to as the ‘leading strand’ and can be created in one continuous go. On the antiparallel strand, the DNA polymersase still attaches at the 3” end and works down towards the 5” end, but this is directly next to the replication form.

Therefore, every time the replication fork unwinds further, the enzyme has to reattach to the 3” end, and this creates small fragments of DNA. This strand is called the ‘lagging strand’ and the small fragments are called Okazaki fragments. The Okazaki fragments are later joined together by the enzyme DNA ligase.

Nucleic Acids, figure 1

Both DNA template strands are now replicated and this process continues until the entire length of DNA is replication.

Nucleic Acids, figure 2

Semi Conservative Replication

DNA replication is described as semi-conservation because in replication one strand is conserved and one new strand is created.

Nucleic Acids, figure 1

Meselson and Stahl performed the experiment below to prove this.

Nucleic Acids, figure 2

Bacteria were grown in a solution containing the 15N isotope. This is a heavier form of nitrogen. During replication, all the new DNA molecules incorporated will contain this isotope in the nitrogenous base. The DNA is therefore heavier, and this is demonstrated by centrifuging and seeing the DNA band settling at a lower point in the test-tube.

A sample of this bacteria is then transferred to a solution containing only the 14N lighter isotope of nitrogen and left to replicate once only. All the DNA newly synthesised will now be lighter. After centrifugation all the DNA settles in the middle of the test-tube, which shows that in DNA replication 50% of the old DNA is always conserved and 50% of the DNA is new.

The bacteria are left to replicate for a second time in the light 14N medium. After another round of semi-conservative replication the results can be seen above. There will now be two double helixes composed of completely light DNA and two double helixes that contain one heavy strand and one light strand.

If the bases on a section of the template DNA strand were TACGGCAAA, what would the bases on the newly formed strand be?
In Meselson and Stahl’s experiment, which part of the DNA nucleotide contains the nitrogen?
Your answer should include: Nitrogenous / base
Name the enzymes that unwinds and unzips the double helix:
Name the enzyme that polymerises the new DNA strand:
Your answer should include: DNA / Polymerase
Name the enzyme that joins the Okazaki fragments together:
Your answer should include: DNA / Ligase
Which strand are the Okazaki fragments located on?
Your answer should include: Lagging / strand
Does DNA polymerase attach to the 3” or the 5” end on the template strand?