Learning Objectives Know how DNA replicates Models of replication Evidence for Semi-Conservative Replication

When cells divide the daughter cell must receive an exact copy of the genetic material from the parent. In order for this to happen the DNA must be replicated or copied.

DNA helicase unzips the 2 strands The enzymes breaks the hydrogen bonds holding the base pairs together.

Free Nucleotides Bind Complementary base pairing DNA helicase completes the splitting of the strand. Meanwhile, free nucleotides that have been activated are attracted to their complementary bases. Each chain acts as a template.

DNA Polymerase Once in placed the activated nucleotides are joined together by DNA polymerase. DNA polymerase joins the new nucleotides to each other by strong covalent bonds, forming the phosphate- sugar backbone.

Replication Finished The result is that there are two DNA molecules, each with one new synthesised strand of DNA and one strand from the original. The DNA is then rewound by another enzyme.

Steps of Replication 1. Replication starts at a specific sequence on the DNA molecule. 2. DNA helicase unwinds and unzips DNA, breaking the hydrogen bonds that join the base pairs, and forming two separate strands. 3. The new DNA is built up from the four nucleotides (A, C, G and T) that are abundant in the nucleoplasm. 4. These nucleotides attach themselves to the bases on the old strands by complementary base pairing. Where there is a T base, only an A nucleotide will bind, and so on. 5. DNA polymerase joins the new nucleotides to each other by strong covalent bonds, forming the phosphate-sugar backbone. 6. A winding enzyme winds the new strands up to form double helices. 7. The two new molecules are identical to the old molecule.

Two theories existed... Conservative Hypothesis 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. Semi-conservative Hypothesis 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).

2 models

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

Meselsohn and Stahl (1958) They grew the bacteria Escherichia coli with different isotopes of Nitrogen. The bacteria was exposed to N 15 for several generations until it was exposed to a lighter N 14. Scientists could then distinguish between the different DNA densities by centrifuging them.

They knew... All the bases in DNA contain Nitrogen Nitrogen has two forms: Light 14 N Heavy isotope 15 N Bacteria will incorporate nitrogen from their growing medium into any new DNA they make

Proving replication is semi-conservative The 15N strand is heavy so makes a band low down the tube High density (heavy) sinks further down the tube. After one generation After one generation there was only one band This was the 14/15 hybrid Second generation After another generation there were 2 bands A light chain and a 14/15 level