DNA replication

DNA replication

DNA replication occurs during the S phase of the cell cycle, illustrated below. This happens within the nucleus in eukaryotic cells. The DNA replication that occurs in all living cells is termed as semiconservative, meaning that the new DNA molecule will have one original strand (also called the parental strand) and one new strand of DNA. This model of DNA replication is most widely accepted, but another model termed conservative replication was also put forward. At the end of this article, we will discuss the evidence as to why semiconservative replication is the accepted model.

Fig. 1 - The phases of the cell cycle

Semiconservative DNA replication steps

Semiconservative replication states that each strand of the original DNA molecule serves as a template for the synthesis of a new DNA strand. The steps for replication outlined below must be accurately executed with high fidelity to prevent the daughter cells from containing mutated DNA, which is DNA that has been replicated incorrectly.

1. The DNA double helix unzips due to the enzyme DNA helicase . This enzyme breaks the hydrogen bonds between the complementary base pairs. A replication fork is created, which is the Y-shaped structure of the DNA unzipping. Each 'branch' of the fork is a single strand of exposed DNA.

2. Free DNA nucleotides in the nucleus will pair with their complementary base on the exposed DNA template strands. Hydrogen bonds will form between the complementary base pairs.

3. The enzyme DNA polymerase forms phosphodiester bonds between adjacent nucleotides in condensation reactions. DNA polymerase binds to the 3 'end of DNA which means the new DNA strand is extending in the 5' to 3 'direction.

Fig. 2 - The semiconservative DNA replication steps

Continuous and discontinuous replication

DNA polymerase, the enzyme which catalyses the formation of phosphodiester bonds, can only make new DNA strands in the 5 'to 3' direction. This strand is called the leading strand and this undergoes continuous replication as it is being continuously synthesized by DNA polymerase, which travels towards the replication fork.

This means the other new DNA strand needs to be synthesized in the 3 'to 5' direction. But how does that work if DNA polymerase travels in the opposite direction? This new strand termed the lagging strand is synthesized in fragments, called Okazaki fragments. Discontinuous replication occurs in this case as DNA polymerase travels away from the replication fork. The Okazaki fragments need to be joined together by phosphodiester bonds and this is catalysed by another enzyme called DNA ligase.

What are the DNA replication enzymes?

Semiconservative DNA replication relies on the action of enzymes. The 3 main enzymes involved are:

• DNA helicase
• DNA polymerase
• DNA ligase

DNA helicase

DNA helicase is involved in the early steps of DNA replication. It breaks the hydrogen bonds between the complementary base pairs to expose the bases on the original strand of DNA. This allows free DNA nucleotides to attach to their complementary pair.

DNA polymerase

DNA polymerase catalyses the formation of new phosphodiester bonds between the free nucleotides in condensation reactions. This creates the new polynucleotide strand of DNA.

DNA ligase

DNA ligase works to join Okazaki fragments together during discontinuous replication through catalyzing the formation of phosphodiester bonds. Although both DNA polymerase and DNA ligase form phosphodiester bonds, both enzymes are needed as they each have different active sites for their specific substrates. DNA ligase is also a key enzyme involved in recombinant DNA technology with plasmid vectors.

Evidence for semiconservative DNA replication

Two models of DNA replication have historically been put forward: conservative and semiconservative DNA replication.

The conservative DNA replication model suggests that after one round, you are left with the original DNA molecule and an entirely new DNA molecule made of new nucleotides. The semiconservative DNA replication model, however, suggests that after one round, the two DNA molecules contain one original strand of DNA and one new strand of DNA. This is the model we explored earlier in this article.

Meselson and Stahl experiment

In the 1950s, two scientists named Matthew Meselson and Franklin Stahl performed an experiment that led to the semiconservative model becoming widely accepted in the scientific community.

So how did they do this? The DNA nucleotides contain nitrogen within the organic bases and Meselson and Stahl knew there were 2 isotopes of nitrogen: N15 and N14, with N15 being the heavier isotopes.

The scientists began by culturing E. coli in a medium containing only N15, which led to the bacteria taking up the nitrogen and incorporating it into their DNA nucleotides. This effectively labelled the bacteria with N15.

The same bacteria were then cultured in a different medium containing only N14 and were allowed to divide over several generations. Meselson and Stahl wanted to measure the DNA density and thus the amount of N15 and N14 in the bacteria so they centrifuged samples after each generation. In the samples, DNA that is lighter in weight will appear higher in the sample tube than DNA that is heavier. These were their results after each generation:

• Generation 0: 1 single band. This indicates the bacteria only contained N15.
• Generation 1: 1 single band in an intermediate position relative to Generation 0 and the N14 control. This indicates that the DNA molecule is made of both N15 and N14 and thus has an intermediate density. The semiconservative DNA replication model predicted this outcome.
• Generation 2: 2 bands with 1 band in the intermediate position which contains both N15 and N14 (like Generation 1) and the other band positioned higher, which contains only N14. This band is positioned higher than N14 has a lower density than N15.

Fig. 3 - Illustration of the findings of the Meselson and Stahl experiment

The evidence from Meselson and Stahl's experiment demonstrates that each DNA strand acts as a template for a new strand and that, after each round of replication, the resulting DNA molecule contains both an original and a new strand. As a result, the scientists concluded that DNA replicates in a semiconservative manner.